Cyclopentane-based human NK1 antagonists. Part 2: development of potent, orally active, water-soluble derivatives.

The synthesis and optimization of a cyclopentane-based hNK1 antagonist scaffold 3, having four chiral centers, will be discussed in the context of its enhanced water solubility properties relative to the marketed anti-emetic hNK1 antagonist EMEND (Aprepitant). Sub-nanomolar hNK1 binding was achieved and oral activity comparable to Aprepitant in two in vivo models will be described.

Transcription regulation of ompF and ompC by a single transcription factor, OmpR.

The ompF and ompC genes of Escherichia coli are reciprocally regulated by a single transcription factor, phosphorylated OmpR (OmpR-P), depending upon medium osmolarity. This regulation involves activation of ompF and its repression with concomitant activation of ompC. This occurs through OmpR-P binding to four (F1, F2, F3, and F4) and three (C1, C2, and C3) sites located upstream of the ompF and ompC promoters, respectively, through a novel mechanism. Here we show that there is a distinct OmpR-P binding hierarchy within F1, F2, and F3 sites as well as within C1, C2, and C3 sites. Each of these sites contains two tandem 10-bp OmpR-P-binding subsites, a-site and b-site (from 5' to 3' direction). OmpR-P has higher affinity to the downstream b-site than to the upstream a-site in each case. Six OmpR-P molecules bind to F and C sites two-by-two in a discontinuous "galloping" manner. We propose that this tight hierarchical binding of a transcription factor, OmpR, allows distinct stepwise regulation of ompF and ompC transcription, which minimizes their overlapping expression upon changes in the medium osmolarity to achieve the reciprocal expression of ompF and ompC.

Bioisosteric replacement of anilide with benzoxazole: potent and orally bioavailable antagonists of VLA-4.

We have designed and synthesized a series of heterocyclic bioisosteres for an anilide based on molecular modeling. Excellent potency was retained in the benzoxazole and the benzimidazole derivatives, where a hydrogen bond acceptor is appropriately positioned to mimic the amide bond oxygen. The deletion of the hydrogen bond donor (N-H) led to improved lipophilicity and bioavailability. In the process, 9a was identified as a potent, specific, and bioavailable VLA-4 antagonist, while 9c was found to be a potent and bioavailable dual antagonist of VLA-4 and alpha(4)beta(7).

A small molecule alpha4beta1/alpha4beta7 antagonist differentiates between the low-affinity states of alpha4beta1 and alpha4beta7: characterization of divalent cation dependence.

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.

N-(3-phenylsulfonyl-3-piperidinoyl)-phenylalanine derivatives as potent, selective VLA-4 antagonists.

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.

Substituted 3-amino biaryl propionic acids as potent VLA-4 antagonists.

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.

N-aryl-prolyl-dipeptides as potent antagonists of VLA-4.

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.

Alpha(4)beta(7)/alpha(4)beta(1) dual integrin antagonists block alpha(4)beta(7)-dependent adhesion under shear flow.

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.

N-Tetrahydrofuroyl-(L)-phenylalanine derivatives as potent VLA-4 antagonists.

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.

Substituted tetrahydrofuroyl-1-phenylalanine derivatives as potent and specific VLA-4 antagonists.

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.

The discovery of acylated beta-amino acids as potent and orally bioavailable VLA-4 antagonists.

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.

Specific and dual antagonists of alpha(4)beta(1) and alpha(4)beta(7) integrins.

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.

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.