Synthesis and SAR studies of novel benzodiazepinedione-based inhibitors of Clostridium difficile (C. difficile) toxin B (TcdB).

Synthesis and structure-activity relationships (SAR) of a novel series of benzodiazepinedione-based inhibitors of Clostridium difficile toxin B (TcdB) are described. Compounds demonstrating low nanomolar affinity for TcdB, and which possess improved stability in mouse plasma vs. earlier compounds from this series, have been identified. Optimized compound 11d demonstrates a good pharmacokinetic (PK) profile in mouse and hamster and is efficacious in a hamster survival model of Clostridium difficile infection.

Treatment of Clostridium difficile Infection with a Small-Molecule Inhibitor of Toxin UDP-Glucose Hydrolysis Activity.

Clostridium difficile infection (CDI) is the leading cause of hospital-acquired infectious diarrhea, with significant morbidity, mortality, and associated health care costs. The major risk factor for CDI is antimicrobial therapy, which disrupts the normal gut microbiota and allows C. difficile to flourish. Treatment of CDI with antimicrobials is generally effective in the short term, but recurrent infections are frequent and problematic, indicating that improved treatment options are necessary. Symptoms of disease are largely due to two homologous toxins, TcdA and TcdB, which are glucosyltransferases that inhibit host Rho GTPases. As the normal gut microbiota is an important component of resistance to CDI, our goal was to develop an effective nonantimicrobial therapy. Here, we report a highly potent small-molecule inhibitor (VB-82252) of TcdA and TcdB. This compound inhibits the UDP-glucose hydrolysis activity of TcdB and protects cells from intoxication after challenge with either toxin. Oral dosing of the inhibitor prevented inflammation in a murine intrarectal toxin challenge model. In a murine model of recurrent CDI, the inhibitor reduced weight loss and gut inflammation during acute disease and did not cause the recurrent disease that was observed with vancomycin treatment. Lastly, the inhibitor demonstrated efficacy similar to that of vancomycin in a hamster disease model. Overall, these results demonstrate that small-molecule inhibition of C. difficile toxin UDP-glucose hydrolysis activity is a promising nonantimicrobial approach to the treatment of CDI.

Identification and initial optimization of inhibitors of Clostridium difficile (C. difficile) toxin B (TcdB).

The discovery, synthesis and preliminary structure-activity relationship (SAR) of a novel class of inhibitors of Clostridium difficile (C. difficile) toxin B (TcdB) is described. A high throughput screening (HTS) campaign resulted in the identification of moderately active screening hits 1-5 the most potent of which was compound 1 (IC50 = 0.77 µM). In silico docking of an early analog offered suggestions for structural modification which resulted in the design and synthesis of highly potent analogs 13j(IC50 = 1 nM) and 13 l(IC50 = 7 nM) which were chosen as leads for further optimization.

Small molecule chemokine mimetics suggest a molecular basis for the observation that CXCL10 and CXCL11 are allosteric ligands of CXCR3.

BACKGROUND AND PURPOSE: The chemokine receptor CXCR3 directs migration of T-cells in response to the ligands CXCL9/Mig, CXCL10/IP-10 and CXCL11/I-TAC. Both ligands and receptors are implicated in the pathogenesis of inflammatory disorders, including atherosclerosis and rheumatoid arthritis. Here, we describe the molecular mechanism by which two synthetic small molecule agonists activate CXCR3. EXPERIMENTAL APPROACH: As both small molecules are basic, we hypothesized that they formed electrostatic interactions with acidic residues within CXCR3. Nine point mutants of CXCR3 were generated in which an acidic residue was mutated to its amide counterpart. Following transient expression, the ability of the constructs to bind and signal in response to natural and synthetic ligands was examined. KEY RESULTS: The CXCR3 mutants D112N, D195N and E196Q were efficiently expressed and responsive in chemotaxis assays to CXCL11 but not to CXCL10 or to either of the synthetic agonists, confirmed with radioligand binding assays. Molecular modelling of both CXCL10 and CXCR3 suggests that the small molecule agonists mimic a region of the '30s loop' (residues 30-40 of CXCL10) which interacts with the intrahelical CXCR3 residue D112, leading to receptor activation. D195 and E196 are located in the second extracellular loop and form putative intramolecular salt bridges required for a CXCR3 conformation that recognizes CXCL10. In contrast, CXCL11 recognition by CXCR3 is largely independent of these residues. CONCLUSION AND IMPLICATIONS: We provide here a molecular basis for the observation that CXCL10 and CXCL11 are allosteric ligands of CXCR3. Such findings may have implications for the design of CXCR3 antagonists.

Identification of a novel allosteric binding site in the CXCR2 chemokine receptor.

We have shown previously that different chemical classes of small-molecule antagonists of the human chemokine CXCR2 receptor interact with distinct binding sites of the receptor. Although an intracellular binding site for diarylurea CXCR2 antagonists, such as N-(2-bromophenyl)-N'-(7-cyano-1H-benzotriazol-4-yl)urea (SB265610), and thiazolopyrimidine compounds was recently mapped by mutagenesis studies, we now report on an imidazolylpyrimidine antagonist binding pocket in the transmembrane domain of CXCR2. Using different CXCR2 orthologs, chimeric proteins, site-directed mutagenesis, and in silico modeling, we have elucidated the binding mode of this antagonist. Our in silico-guided mutagenesis studies indicate that the ligand binding cavity for imidazolylpyrimidine compounds in CXCR2 is located between transmembrane (TM) helices 3 (Phe130(3.36)), 5 (Ser217(5.44), Phe220(5.47)), and 6 (Asn268(6.52), Leu271(6.55)) and suggest that these antagonists enter CXCR2 via the TM5-TM6 interface. It is noteworthy that the same interface is postulated as the ligand entry channel in the opsin receptor and is occupied by lipid molecules in the recently solved crystal structure of the CXCR4 chemokine receptor, suggesting a general ligand entrance mechanism for nonpolar ligands to G protein-coupled receptors. The identification of a novel allosteric binding cavity in the TM domain of CXCR2, in addition to the previously identified intracellular binding site, shows the diversity in ligand recognition mechanisms by this receptor and offers new opportunities for the structure-based design of small allosteric modulators of CXCR2 in the future.

Synthesis and SAR studies of novel 2-(6-aminomethylaryl-2-aryl-4-oxo-quinazolin-3(4H)-yl)acetamide vasopressin V1b receptor antagonists.

Synthesis and structure-activity relationships (SAR) of a novel series of vasopressin V(1b) antagonists are described. 2-(6-Aminomethylaryl-2-aryl-4-oxo-quinazolin-3(4H)-yl)acetamide have been identified with low nanomolar affinity for the V(1b) receptor and good selectivity with respect to related receptors V(1a), V(2) and OT. Optimised compound 16 shows a good pharmacokinetic profile and activity in a mechanistic model of HPA dysfunction.

Synthesis and SAR studies of novel 2-(4-oxo-2-aryl-quinazolin-3(4H)-yl)acetamide vasopressin V1b receptor antagonists.

Synthesis and structure-activity relationships (SAR) of a novel series of vasopressin V(1b) (V(3)) antagonists are described. 2-(4-Oxo-2-aryl-quinazolin-3(4H)-yl)acetamides have been identified with low nanomolar affinity for the V(1b) receptor and good selectivity with respect to related receptors V(1a), V(2) and oxytocin (OT). Optimised compound 12j demonstrates a good pharmacokinetic profile and activity in a mechanistic model of HPA dysfunction.

Novel pyrrolidine heterocycles as CCR1 antagonists.

A novel series of pyrrolidine heterocycles was prepared and found to show potent inhibitory activity of CCR1 binding and CCL3 mediated chemotaxis of a CCR1-expressing cell line. A potent, optimized triazole lead from this series was found to have acceptable pharmacokinetics and microsomal stability in rat and is suitable for further optimization and development.

Identification and optimization of novel 2-(4-oxo-2-aryl-quinazolin-3(4H)-yl)acetamide vasopressin V3 (V1b) receptor antagonists.

The discovery, synthesis, and preliminary structure-activity relationship (SAR) of a novel class of vasopressin V3 (V1b) receptor antagonists is described. Compound 1, identified by high throughput screening of a diverse, three million-member compound collection, prepared using ECLiPS technology, had good activity in a V3 binding assay (IC50=0.20 microM), but less than desirable physicochemical properties. Optimization of compound 1 yielded potent analogs 19 (IC50=0.31 microM) and 24 (IC50=0.12 microM) with improved drug-like characteristics.

Identification and hit-to-lead optimization of a novel class of CB1 antagonists.

The discovery, synthesis and preliminary structure-activity relationships (SARs) of a novel class of CB1 antagonists is described. Initial optimization of benzimidazole-based screening hit 4 led to the identification of 'inverted' indole-based lead compound 18c with improved properties versus compound 4 including reduced AlogP, improved microsomal stability and improved aqueous solubility. Compound 18c demonstrates in vivo CB1 antagonist efficacy (CB1 agonist induced hypothermia model) and is orally bioavailable in rat.

Selective functional inhibition of JAK-3 is sufficient for efficacy in collagen-induced arthritis in mice.

OBJECTIVE: All gamma-chain cytokines signal through JAK-3 and JAK-1 acting in tandem. We undertook this study to determine whether the JAK-3 selective inhibitor WYE-151650 would be sufficient to disrupt cytokine signaling and to ameliorate autoimmune disease pathology without inhibiting other pathways mediated by JAK-1, JAK-2, and Tyk-2. METHODS: JAK-3 kinase selective compounds were characterized by kinase assay and JAK-3-dependent (interleukin-2 [IL-2]) and -independent (IL-6, granulocyte-macrophage colony-stimulating factor [GM-CSF]) cell-based assays measuring proliferation or STAT phosphorylation. In vivo, off-target signaling was measured by IL-22- and erythropoietin (EPO)-mediated models, while on-target signaling was measured by IL-2-mediated signaling. Efficacy of JAK-3 inhibitors was determined using delayed-type hypersensitivity (DTH) and collagen-induced arthritis (CIA) models in mice. RESULTS: In vitro, WYE-151650 potently suppressed IL-2-induced STAT-5 phosphorylation and cell proliferation, while exhibiting 10-29-fold less activity against JAK-3-independent IL-6- or GM-CSF-induced STAT phosphorylation. Ex vivo, WYE-151650 suppressed IL-2-induced STAT phosphorylation, but not IL-6-induced STAT phosphorylation, as measured in whole blood. In vivo, WYE-151650 inhibited JAK-3-mediated IL-2-induced interferon-gamma production and decreased the natural killer cell population in mice, while not affecting IL-22-induced serum amyloid A production or EPO-induced reticulocytosis. WYE-151650 was efficacious in mouse DTH and CIA models. CONCLUSION: In vitro, ex vivo, and in vivo assays demonstrate that WYE-151650 is efficacious in mouse CIA despite JAK-3 selectivity. These data question the need to broadly inhibit JAK-1-, JAK-2-, or Tyk-2-dependent cytokine pathways for efficacy.

2-Benzimidazolyl-9-(chroman-4-yl)-purinone derivatives as JAK3 inhibitors.

A novel class of Janus tyrosine kinase 3 (JAK3) inhibitors based on a 2-benzimidazoylpurinone core structure is described. Through substitution of the benzimidazoyl moiety and optimization of the N-9 substituent of the purinone, compound 24 was identified incorporating a chroman-based functional group. Compound 24 shows excellent kinase activity, good oral bioavailability and demonstrates efficacy in an acute mechanistic mouse model through inhibition of interleukin-2 (IL-2) induced interferon-gamma (INF-gamma) production.

Novel pyrrolidine ureas as C-C chemokine receptor 1 (CCR1) antagonists.

Monocyte infiltration is implicated in a variety of diseases including multiple myeloma, rheumatoid arthritis, and multiple sclerosis. C-C chemokine receptor 1 (CCR1) is a chemokine receptor that upon stimulation, particularly by macrophage inflammatory protein 1alpha (MIP-1alpha) and regulated on normal T-cell expressed and secreted (RANTES), mediates monocyte trafficking to sites of inflammation. High throughput screening of our combinatorial collection identified a novel, moderately potent CCR1 antagonist 3. The library hit 3 was optimized to the advanced lead compound 4. Compound 4 inhibited CCR1 mediated chemotaxis of monocytes with an IC(50) of 20 nM. In addition, the compound was highly selective over other chemokine receptors. It had good microsomal stability when incubated with rat and human liver microsomes and showed no significant cytochrome P450 (CYP) inhibition. Pharmacokinetic evaluation of the compound in the rat showed good oral bioavailability.

Synthesis and initial evaluation of novel, non-peptidic antagonists of the alpha(v)-integrins alpha(v)beta(3) and alpha(v)beta(5).

The discovery, synthesis and preliminary SAR of a novel class of non-peptidic antagonists of the alpha(v)-integrins alpha(v)beta(3) and alpha(v)beta(5) is described. High-throughput screening of an extensive series of ECLiPStrade mark compound libraries led to the identification of compound 1 as a dual inhibitor of the alpha(v)-integrins alpha(v)beta(3) and alpha(v)beta(5). Optimization of compound 1 involving, in part, introduction of two novel constraints led to the discovery of compounds 15a and 15b with reduced PSA and much improved potency for both the alpha(v)beta(3) and alpha(v)beta(5) integrins. Compounds 15a and 15b were shown to have promising activity in functional cellular assays and compound 15a also exhibited a promising Caco-2 permeability profile.

Synthesis of (3,4-dimethoxyphenoxy)alkylamino acetamides as orexin-2 receptor antagonists.

The discovery and synthesis of a series of (dimethoxyphenoxy)alkylamino acetamides as orexin-2 receptor antagonists from a small-molecule combinatorial library using a high-throughput calcium mobilization functional assay (HEK293-human OX2-R cell line) is described. Active compounds show a good correlation between high-throughput single concentration screening data and measured IC(50)s. Specific examples exhibit IC(50) values of approximately 20 nM using human orexin A as the peptide agonist for the orexin-2 receptor.

Kinase inhibitors as drugs for chronic inflammatory and immunological diseases: progress and challenges.

At the time of writing, there are seven marketed kinase inhibitor drugs. The first kinase inhibitor, imatinib mesilate (Gleevec, Novartis), came to market in 2001, an inhibitor of the breakpoint cluster region (BCR)/Abelson murine leukemia oncogene homolog (ABL) fusion, platelet-derived growth factor (PDGF) receptor, and c-kit kinases. The most recent kinase inhibitor to come to market, disatinib (Sprycel, Bristol-Myers Squibb), acts on c-SRC, ABL and Bruton's tyrosine kinase. To date, kinase inhibitor drugs are approved for oncology and demonstrate that it is possible to develop compounds with relative selectivity for the target kinase against the broader kinome. However, the use of kinase inhibitors in chronic inflammatory and immunologic diseases may require greater selectivity for the target kinase. This review addresses the opportunities and challenges of kinase inhibition as a therapeutic approach in chronic immune and inflammatory disease.

Identification and characterization of small-molecule inhibitors of Tie2 kinase.

Angiopoietins and Tie2 receptor were recently identified as an endothelial cell-specific ligand-receptor system that is critical for vascular development and postnatal pathologic angiogenesis by mediating vascular integrity. In this study, we identified a series of small-molecule Tie2 inhibitors, which blocked Ang1-induced Tie2 autophosphorylation and downstream signaling with an IC(50) value at 0.3 microM. Further optimization yields improved selectivity, aqueous solubility, microsomal stability and cytochrome P450 profile for one of the compounds (compound 7). Both compound 1 and compound 7 inhibit endothelial cell tube formation. Furthermore, in a rat model of Matrigel-induced choroidal neovascularization, compound 7 significantly diminished aberrant vessel growth. Our findings demonstrate a potential clinical benefit by specifically targeting Tie2-mediated angiogenic disorders.

Discovery and preliminary evaluation of 5-(4-phenylbenzyl)oxazole-4-carboxamides as prostacyclin receptor antagonists.

The discovery and evaluation of 5-(4-phenylbenzyl)oxazole-4-carboxamides as prostacyclin (IP) receptor antagonists is described. Analogs disclosed showed high affinity for the IP receptor in human platelet membranes with IC50 values of 0.05-0.50 microM, demonstrated functional antagonism by inhibiting cAMP production in HEL cells with IC50 values of 0.016-0.070 microM, and exhibited significant selectivity versus other prostanoid receptors.

Identification of CXCR3 receptor agonists in combinatorial small-molecule libraries.

In a high-throughput screen of four million compounds from combinatorial libraries for small-molecule modulators of the chemokine receptor CXCR3, two classes of receptor agonists, based on tetrahydroisoquinoline and piperidinyl diazepanone templates, were identified. Several of these compounds stimulated calcium flux in HEK293 cells expressing the recombinant human CXCR3 receptor with efficacies and kinetics similar to those of native ligand CXCL11/I-TAC and stimulated chemotaxis of activated human T-cells. The agonist small molecules also inhibited binding of another CXCR3 ligand, CXCL10/IP-10, to the receptor. The response to small-molecule agonists was inhibited by a CXCR3-specific small-molecule antagonist previously identified within the same combinatorial compound collection but structurally unrelated to the agonists. Remarkably, while other, non-amino acid substituents were present in the majority of the library compounds screened, the agonists from both classes contained a positively charged amino acid component, with preference for Arg>Lys, as well as a hydrophobic component.