Tricyclic pharmacophore-based molecules as novel integrin alpha(v)beta3 antagonists. Part 2: synthesis of potent alpha(v)beta3/alpha(IIb)beta3 dual antagonists

We synthesized 4-aminopiperidine derivatives of our prototype integrin alpha(v)beta3 antagonist 1 in an attempt to increase the activity and water solubility. Introduction of one or two hydrophilic moieties into the central aromatic ring and/or the benzene ring at the C-terminus of 1 increased water solubility and enhanced inhibition of cell adhesion. The results of a structure-activity relationships (SAR) study indicated that the torsion angle between the central aromatic ring and the piperidine ring, and the acidity at the sulfonamide moiety, might be important for alpha(v)beta3 receptor binding activity. Some of these compounds are novel and potent alpha(v)beta3/alpha(IIb)beta3 dual antagonists with acceptable water solubility and a satisfactory early absorption, distribution, metabolism, excretion, and toxicity (ADMET) profile.     

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.     

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.     

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.     

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.     

Convergent, parallel synthesis of a series of beta-substituted 1,2,4-oxadiazole butanoic acids as potent and selective alpha(v)beta3 receptor antagonists

We describe a series of 1,2,4-oxadiazoles, which are potent antagonists of the integrin alpha(v)beta3 and, in addition, show selectivity relative to the other beta3 integrin alpha(IIb)beta3. In whole cells, the majority of these analogs also demonstrated modest selectivity against other alpha(v) integrins such as alpha(v)beta1 and alpha(v)beta6.     

Structural requirements of echistatin for the recognition of alpha(v)beta(3) and alpha(5)beta(1) integrins

There are key differences between the amino acid residues of the RGD loops and the C termini of echistatin, a potent antagonist of alpha(IIb)beta(3), alpha(v)beta(3) and alpha(5)beta(1), and eristostatin, a similar disintegrin selectively inhibiting alpha(IIb)beta(3). In order to identify echistatin motifs required for selective recognition of alpha(v)beta(3) and alpha(5)beta(1) integrins, we expressed recombinant echistatin, eristostatin, and 15 hybrid molecules. We tested them for their ability to inhibit adhesion of different cell lines to fibronectin and von Willebrand factor and to express ligand-induced binding site epitope. The results showed that Asp(27) and Met(28) support recognition of both alpha(v)beta(3) and alpha(5)beta(1). Replacement of Met(28) with Asn completely abolished echistatin's ability to recognize each of the integrins, while replacement of Met(28) with Leu selectively decreased echistatin's ability to recognize alpha(5)beta(1) only. Eristostatin in which C-terminal WNG sequence was substituted with HKGPAT exhibited new activity with alpha(5)beta(1), which was 10-20-fold higher than that of wild type eristostatin. A hypothesis is proposed that the C terminus of echistatin interacts with separate sites on beta(1) and beta(3) integrin molecules.     

RGD mimetics containing a central hydantoin scaffold: alpkha(v)beta3 vs alpha(IIb)beta3 selectivity requirements

The synthesis of a series of RGD mimetic alpha(v)beta3 antagonists containing a hydantoin scaffold is shown. The results demonstrate some of the structural requirements for the design of selective alpha(v)beta3 antagonists (vs alpha(IIb)beta3) in terms of the Arg-mimetic, the distance between N- and C-terminus and the lipophilic side chain.     

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.     

Synthesis and SAR of N-substituted dibenzazepinone derivatives as novel potent and selective alpha(V)beta(3) antagonists

Synthesis and SARs of new integrin alpha(V)beta(3) antagonists based on an N-substituted dibenzazepinone scaffold are described. Variation of spacer and guanidine mimetic led to potent compounds exhibiting an IC(50) towards alpha(V)beta(3) in the nanomolar range, high selectivity versus integrin alpha(IIb)beta(3) and efficacy in functional cellular assays.     

alpha(v)beta(3) Antagonists based on a central thiophene scaffold.

A series of novel, highly potent alpha(v)beta(3) antagonists based on a thiophene scaffold and containing an acylguanidine as an Arg-mimetic is described. A number of structural features, such as cyclic versus open guanidine and a variety of lipophilic side chains, carbamates, sulfonamides and beta-amino acids were explored with respect to inhibition of alpha(v)beta(3) mediated cell adhesion and selectivity versus alpha(IIb)beta(3) binding. In addition, compound 19 was found to be active in the TPTX model of osteoporosis.     

Non-peptidic alpha(v)beta3 antagonists containing indol-1-yl propionic acids

We describe the synthesis and structure/activity relationship of RGD mimetics that are potent inhibitors of the integrin alpha(v)beta3. Indol-1-yl propionic acids containing a variety of basic moieties at the 5-position, as well as substitutions alpha and beta to the carboxy terminus were synthesized and evaluated. Novel compounds with improved potency have been identified.     

Synthesis of cinnamic acids and related isosteres as potent and selective alpha v beta 3 receptor antagonists

We describe a series of conformationally-restricted cinnamic acid peptidomimetics as well as several cinnamic acid isosteres, including 3-phenylpropionic acids, 2-amino-3-phenylpropionic acids, phenoxyacetic acids and 2-phenylcyclopropylcarboxylic acids. Several analogues demonstrated low to sub-nanomolar potencies against alpha(v)beta(3) and greater than 200-fold selectivity against the other beta(3) integrin alpha(IIb)beta(3). In whole 293 cells, many of these analogues also showed modest selectivity against other alpha(v) integrins such as alpha(v)beta(1) and alpha(v)beta(5). These compounds were synthesized from readily available starting materials using either Heck or Mitsunobu coupling conditions.     

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.     

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.     

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.     

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.     

Evidence for a differential functional regulation of the two beta(3)-integrins alpha(V)beta(3) and alpha(IIb)beta(3)

The functional regulation of integrins is a major determinant of cell adhesion, migration and tissue maintenance. The binding of cytoskeletal proteins to various sites of integrin cytoplasmic domains is a key mechanism of this functional regulation. Expression of recombinant integrin alpha(IIb)beta(3) and alpha(M)beta(2) lacking the GFFKR-region in CHO cells results in constitutively activated integrins. In contrast, CHO cells stably expressing either a GFFKR-deleted alpha(V(del))beta(3) or a FF to AA-substituted alpha(V(AA))beta(3) do not reveal a constitutively activated integrin. Adhesion to immobilized fibrinogen is strongly impaired in alpha(V(del))beta(3) or alpha(V(AA))beta(3)-expressing cells, whereas it is not impaired in alpha(IIb)beta(3) and alpha(M)beta(2), both lacking the GFFKR-region. In a parallel plate flow chamber assay, alpha(V)beta(3)-expressing cells adhere firmly to fibrinogen and spread even at shear rates of 15 to 20 dyn/cm(2), whereas alpha(V(del))beta(3) or alpha(V(AA))beta(3) cells are detached at 15 dyn/cm(2). Actin stress fiber formation and focal adhesion plaques containing alpha(V)beta(3) are observed in alpha(V)beta(3) cells but not in alpha(V(del))beta(3) or alpha(V(AA))beta(3)-expressing cells. As an additional manifestation of impaired outside-in signaling, phosphorylation of pp125(FAK) was reduced in these cells. In summary, we report that the GFFKR-region of the alpha(V)-cytoplasmic domain and in particular two phenylalanines are essential for integrin alpha(V)beta(3) function, especially for outside-in signaling. Our results suggest that the two beta(3)-integrins alpha(IIb)beta(3) and alpha(V)beta(3) are differentially regulated via their GFFKR-region.     

Aza-bicyclic amino acid sulfonamides as alpha(4)beta(1)/alpha(4)beta(7) integrin antagonists

The design, synthesis, and biological activity of novel alpha(4)beta(1) and alpha(4)beta(7) integrin antagonists, containing a bridged azabicyclic nucleus, are reported. Conformational analysis of targets containing an azabicyclo[2.2.2]octane carboxylic acid and known integrin antagonists indicated that this azabicycle would be a suitable molecular scaffold. Variation of substituents on the pendant arylsulfonamide and phenylalanine groups resulted in potent alpha(4)beta(1)-selective and dual alpha(4)beta(1)/alpha(4)beta(7) antagonists. Potent compounds 11i, 11h, and 14 were effective in the antigen-sensitized sheep model of asthma.