Design, synthesis, and activity of 2-imidazol-1-ylpyrimidine derived inducible nitric oxide synthase dimerization inhibitors.

By the screening of a combinatorial library for inhibitors of nitric oxide (NO) formation by the inducible isoform of nitric oxide synthase (iNOS) using a whole-cell assay, 2-(imidazol-1-yl)pyrimidines were identified. Compounds were found to inhibit the dimerization of iNOS monomers, thus preventing the formation of the dimeric, active form of the enzyme. Optimization led to the selection of the potent, selective, and orally available iNOS dimerization inhibitor, 21b, which significantly ameliorated adjuvant-induced arthritis in a rat model. Analysis of the crystal structure of the 21b--iNOS monomer complex provided a rationalization for both the SAR and the mechanism by which 21b blocks the formation of the protein--protein interaction present in the dimeric form of iNOS.

Thiophene-anthranilamides as highly potent and orally available factor Xa inhibitors.

There remains a high unmet medical need for a safe oral therapy for thrombotic disorders. The serine protease factor Xa (fXa), with its central role in the coagulation cascade, is among the more promising targets for anticoagulant therapy and has been the subject of intensive drug discovery efforts. Investigation of a hit from high-throughput screening identified a series of thiophene-substituted anthranilamides as potent nonamidine fXa inhibitors. Lead optimization by incorporation of hydrophilic groups led to the discovery of compounds with picomolar inhibitory potency and micromolar in vitro anticoagulant activity. Based on their high potency, selectivity, oral pharmacokinetics, and efficacy in a rat venous stasis model of thrombosis, compounds ZK 814048 (10b), ZK 810388 (13a), and ZK 813039 (17m) were advanced into development.

Human factor Xa bound amidine inhibitor conformation by double rotational-echo double resonance nuclear magnetic resonance and molecular dynamics simulations.

Double rotational-echo double resonance (double REDOR) NMR was used to investigate the conformation of a (13)C-, (15)N-, and (19)F-labeled inhibitor (Berlex Biosciences compound no. ZK-806299) bound to human factor Xa. Conformationally dependent carbon-fluorine dipolar couplings were measured by (13)C[(19)F] REDOR. Natural abundance carbon signals in the full-echo spectra were removed by (13)C[(15)N] REDOR. Major and minor binding modes were suggested by the NMR data, but only the former had adequate signal to noise for distance determinations. Molecular dynamics simulations restrained by double-REDOR-determined intramolecular (13)C-(19)F distances revealed two models for the dominant binding mode that are consistent with the NMR data. We conclude that ZK-806299 binds similarly to both FXa. Moreover, it appears to bind to FXa in a fashion previously demonstrated for ZK-807834, a more selective FXa inhibitor.

Potent triazolothione inhibitor of heat-shock protein-90.

Heat-shock protein-90 is an attractive target for anticancer drugs, as heat-shock protein-90 blockers such as the ansamycin 17-(allylamino)-17-demethoxygeldanamycin greatly reduce the expression of many signaling molecules that are disregulated in cancer cells and are key drivers of tumor growth and metastasis. While 17-(allylamino)-17-demethoxygeldanamycin has shown promise in clinical trials, this compound class has significant template-related drawbacks. In this paper, we describe a new, potent non-ansamycin small-molecule inhibitor of heat-shock protein-90, BX-2819, containing resorcinol and triazolothione rings. Structural studies demonstrate binding of BX-2819 to the ADP/ATP-binding pocket of heat-shock protein-90. The compound blocked expression of heat-shock protein-90 client proteins in cancer cell lines and inhibited cell growth with a potency similar to 17-(allylamino)-17-demethoxygeldanamycin. In a panel of four cancer cell lines, BX-2819 blocked growth with an average IC(50) value of 32 nM (range of 7-72 nM). Efficacy studies demonstrated that treatment with BX-2819 significantly inhibited the growth of NCI-N87 and HT-29 tumors in nude mice, consistent with pharmacodynamic studies showing inhibition of heat-shock protein-90 client protein expression in tumors for greater than 16 h after dosing. These data support further studies to assess the potential of BX-2819 and related analogs for the treatment of cancer.

CCR5 receptor antagonists: discovery and SAR study of guanylhydrazone derivatives.

High throughput screening (HTS) led to the identification of the guanylhydrazone of 2-(4-chlorobenzyloxy)-5-bromobenzaldehyde as a CCR5 receptor antagonist. Initial modifications of the guanylhydrazone series indicated that substitution of the benzyl group at the para-position was well tolerated. Substitution at the 5-position of the central phenyl ring was critical for potency. Replacement of the guanylhydrazone group led to the discovery of a novel series of CCR5 antagonists.

Indolinone based phosphoinositide-dependent kinase-1 (PDK1) inhibitors. Part 2: optimization of BX-517.

Based on the lead compound BX-517, a series of C-4' substituted indolinones have been synthesized and evaluated for PDK1 inhibition. Modification at C-4' of the pyrrole afforded potent compounds (7b and 7d) with improved solubility and ADME properties. In this letter, we describe the synthesis, selectivity profile, and pharmacokinetic data of selected compounds.

Indolinone based phosphoinositide-dependent kinase-1 (PDK1) inhibitors. Part 1: design, synthesis and biological activity.

HTS screening identified 1 with micromolar inhibitory activity against PDK1. Optimization of 1 afforded 4i (BX-517) which has single-digit nanomolar activity against PDK1 and excellent selectivity against PKA.

Novel small molecule inhibitors of 3-phosphoinositide-dependent kinase-1.

The phosphoinositide 3-kinase/3-phosphoinositide-dependent kinase 1 (PDK1)/Akt signaling pathway plays a key role in cancer cell growth, survival, and tumor angiogenesis and represents a promising target for anticancer drugs. Here, we describe three potent PDK1 inhibitors, BX-795, BX-912, and BX-320 (IC(50) = 11-30 nm) and their initial biological characterization. The inhibitors blocked PDK1/Akt signaling in tumor cells and inhibited the anchorage-dependent growth of a variety of tumor cell lines in culture or induced apoptosis. A number of cancer cell lines with elevated Akt activity were >30-fold more sensitive to growth inhibition by PDK1 inhibitors in soft agar than on tissue culture plastic, consistent with the cell survival function of the PDK1/Akt signaling pathway, which is particularly important for unattached cells. BX-320 inhibited the growth of LOX melanoma tumors in the lungs of nude mice after injection of tumor cells into the tail vein. The effect of BX-320 on cancer cell growth in vitro and in vivo indicates that PDK1 inhibitors may have clinical utility as anticancer agents.

The discovery of fluoropyridine-based inhibitors of the Factor VIIa/TF complex.

The activated Factor VII/tissue factor complex (FVIIa/TF) plays a key role in the formation of blood clots. Inhibition of this complex may lead to new antithrombotic drugs. An X-ray crystal structure of a fluoropyridine-based FVIIa/TF inhibitor bound in the active site of the enzyme complex suggested that incorporation of substitution at the 5-position of the hydroxybenzoic acid side chain could lead to the formation of more potent inhibitors through interactions with the S1'/S2' pocket.

Conformation of a bound inhibitor of blood coagulant factor Xa.

13C[(15)N] and (13)C[(19)F] rotational-echo double-resonance NMR have been used to characterize the enzyme-bound structure of ZK-816042, an amidine-imidazoline inhibitor of human factor Xa (FXa). The NMR experiments were performed on a lyophilized FXa-inhibitor complex. The complex was formed in solution in the presence of stabilizing excipients and frozen after gradual supercooling prior to lyophilization. The results indicate that the inhibitor binds with a distribution of orientations of the imidazoline ring.