Identification of novel azaindazole CCR1 antagonist clinical candidates.

Exploring various cyclization strategies, using a submicromolar pyrazole HTS screening hit 6 as a starting point, a novel indazole based CCR1 antagonist core was discovered. This report presents the design and SAR of CCR1 indazole and azaindazole antagonists leading to the identification of three development compounds, including 19e that was advanced to early clinical trials.

Late-stage optimization of a tercyclic class of S1P3-sparing, S1P1 receptor agonists.

Poor solubility and cationic amphiphilic drug-likeness were liabilities identified for a lead series of S1P3-sparing, S1P1 agonists originally developed from a high-throughput screening campaign. This work describes the subsequent optimization of these leads by balancing potency, selectivity, solubility and overall molecular charge. Focused SAR studies revealed favorable structural modifications that, when combined, produced compounds with overall balanced profiles. The low brain exposure observed in rat suggests that these compounds would be best suited for the potential treatment of peripheral autoimmune disorders.

G protein-coupled bile acid receptor 1 stimulation mediates arterial vasodilation through a K(Ca)1.1 (BK(Ca))-dependent mechanism.

Bile acids (BAs) and BA receptors, including G protein-coupled bile acid receptor 1 (GPBAR1), represent novel targets for the treatment of metabolic and inflammatory disorders. However, BAs elicit myriad effects on cardiovascular function, although this has not been specifically ascribed to GPBAR1. This study was designed to test whether stimulation of GPBAR1 elicits effects on cardiovascular function that are mechanism based that can be identified in acute ex vivo and in vivo cardiovascular models, to delineate whether effects were due to pathways known to be modulated by BAs, and to establish whether a therapeutic window between in vivo cardiovascular liabilities and on-target efficacy could be defined. The results demonstrated that the infusion of three structurally diverse and selective GPBAR1 agonists produced marked reductions in vascular tone and blood pressure in dog, but not in rat, as well as reflex tachycardia and a positive inotropic response, effects that manifested in an enhanced cardiac output. Changes in cardiovascular function were unrelated to modulation of the levothyroxine/thyroxine axis and were nitric oxide independent. A direct effect on vascular tone was confirmed in dog isolated vascular rings, whereby concentration-dependent decreases in tension that were tightly correlated with reductions in vascular tone observed in vivo and were blocked by iberiotoxin. Compound concentrations in which cardiovascular effects occurred, both ex vivo and in vivo, could not be separated from those necessary for modulation of GPBAR1-mediated efficacy, resulting in project termination. These results are the first to clearly demonstrate direct and potent peripheral arterial vasodilation due to GPBAR1 stimulation in vivo through activation of large conductance Ca(2+) activated potassium channel K(Ca)1.1.

Substituted phenyl as a steroid A-ring mimetic: providing agonist activity to a class of arylsulfonamide nonsteroidal glucocorticoid ligands.

A class of arylsulfonamide glucocorticoid receptor agonists that contains a substituted phenyl group as a steroid A-ring mimetic is reported. The structural design and SAR that provide the functional switching of a GR antagonist to an agonist is described. A combination of specific hydrogen bonding and lipophilic elements on the A-ring moiety is required to achieve potent GR agonist activity. This study culminated in the identification of compound 23 as a potent GR agonist with selectivity over the PR and MR nuclear hormone receptors.

Function-regulating pharmacophores in a sulfonamide class of glucocorticoid receptor agonists.

A class of α-methyltryptamine sulfonamide glucocorticoid receptor (GR) modulators was optimized for agonist activity. The design of ligands was aided by molecular modeling, and key function-regulating pharmacophoric points were identified that are critical in achieving the desired agonist effect in cell based assays. Compound 27 was profiled in vitro and in vivo in models of inflammation. Analogs could be rapidly prepared in a parallel approach from aziridine building blocks.

Non-steroidal dissociated glucocorticoid agonists: indoles as A-ring mimetics and function-regulating pharmacophores.

We report a SAR of non-steroidal glucocorticoid mimetics that utilize indoles as A-ring mimetics. Detailed SAR is discussed with a focus on improving PR and MR selectivity, GR agonism, and in vitro dissociation profile. SAR analysis led to compound (R)-33 which showed high PR and MR selectivity, potent agonist activity, and reduced transactivation activity in the MMTV and aromatase assays. The compound is equipotent to prednisolone in the LPS-TNF model of inflammation. In mouse CIA, at 30 mg/kg compound (R)-33 inhibited disease progression with an efficacy similar to the 3 mg/kg dose of prednisolone.

Quinol-4-ones as steroid A-ring mimetics in nonsteroidal dissociated glucocorticoid agonists.

We report on the nuclear receptor binding affinities, cellular activities of transrepression and transactivation, and anti-inflammatory properties of a quinol-4-one and other A-ring mimetic containing nonsteroidal class of glucocorticoid agonists.

Second-generation lymphocyte function-associated antigen-1 inhibitors: 1H-imidazo[1,2-alpha]imidazol-2-one derivatives.

A novel class of lymphocyte function-associated antigen-1 (LFA-1) inhibitors is described. Discovered during the process to improve the physicochemical and metabolic properties of BIRT377 (1, Figure 1), a previously reported hydantoin-based LFA-1 inhibitor, these compounds are characterized by an imidazole-based 5,5-bicyclic scaffold, the 1,3,3-trisubstituted 1H-imidazo[1,2-alpha]imidazol-2-one (i.e. structure 3). The structure-activity relationship (SAR) shows that electron-withdrawing groups at C5 on the imidazole ring benefit potency and that oxygen-containing functional groups attached to a C5-sulfonyl or sulfonamide group further improve potency. This latter gain in potency is attributed to the interaction(s) of the functionalized sulfonyl/sulfonamide groups with the protein, likely polar-polar in nature, as suggested by SAR data. X-ray studies revealed that these bicyclic inhibitors bind to the I-domain of LFA-1 in a pattern similar to that of compound 1.

A GPBAR1 (TGR5) small molecule agonist shows specific inhibitory effects on myeloid cell activation in vitro and reduces experimental autoimmune encephalitis (EAE) in vivo.

GPBAR1 is a G protein-coupled receptor that is activated by certain bile acids and plays an important role in the regulation of bile acid synthesis, lipid metabolism, and energy homeostasis. Recent evidence suggests that GPBAR1 may also have important effects in reducing the inflammatory response through its expression on monocytes and macrophages. To further understand the role of GPBAR1 in inflammation, we generated a novel, selective, proprietary GPBAR1 agonist and tested its effectiveness at reducing monocyte and macrophage activation in vitro and in vivo. We have used this agonist, together with previously described agonists to study agonism of GPBAR1, and shown that they can all induce cAMP and reduce TLR activation-induced cytokine production in human monocytes and monocyte-derived macrophages in vitro. Additionally, through the usage of RNA sequencing (RNA-Seq), we identified a select set of genes that are regulated by GPBAR1 agonism during LPS activation. To further define the in vivo role of GPBAR1 in inflammation, we assessed GPBAR1 expression and found high levels on circulating mouse monocytes. Agonism of GPBAR1 reduced LPS-induced cytokine production in mouse monocytes ex vivo and serum cytokine levels in vivo. Agonism of GPBAR1 also had profound effects in the experimental autoimmune encephalomyelitis (EAE) mouse model of multiple sclerosis, where monocytes play an important role. Mice treated with the GPBAR1 agonist exhibited a significant reduction in the EAE clinical score which correlated with reduced monocyte and microglial activation and reduced trafficking of monocytes and T cells into the CNS. These data confirm the importance of GPBAR1 in controlling monocyte and macrophage activation in vivo and support the rationale for selective agonists of GPBAR1 in the treatment of inflammatory diseases.

Identification of highly efficacious glucocorticoid receptor agonists with a potential for reduced clinical bone side effects.

Synthesis and structure-activity relationship (SAR) of a series of nonsteroidal glucocorticoid receptor (GR) agonists are described. These compounds contain "diazaindole" moieties and display different transcriptional regulatory profiles in vitro and are considered "dissociated" between gene transrepression and transactivation. The lead optimization effort described in this article focused in particular on limiting the transactivation of genes which result in bone side effects and these were assessed in vitro in MG-63 osteosarcoma cells, leading to the identification of (R)-18 and (R)-21. These compounds maintained anti-inflammatory activity in vivo in collagen induced arthritis studies in mouse but had reduced effects on bone relevant parameters compared to the widely used synthetic glucocorticoid prednisolone 2 in vivo. To our knowledge, we are the first to report on selective glucocorticoid ligands with reduced bone loss in a preclinical in vivo model.

CCR1 plays a critical role in modulating pain through hematopoietic and non-hematopoietic cells.

Inflammation is associated with immune cells infiltrating into the inflammatory site and pain. CC chemokine receptor 1 (CCR1) mediates trafficking of leukocytes to sites of inflammation. However, the contribution of CCR1 to pain is incompletely understood. Here we report an unexpected discovery that CCR1-mediated trafficking of neutrophils and CCR1 activity on non-hematopoietic cells both modulate pain. Using a genetic approach (CCR1-/- animals) and pharmacological inhibition of CCR1 with selective inhibitors, we show significant reductions in pain responses using the acetic acid-induced writhing and complete Freund's adjuvant-induced mechanical hyperalgesia models. Reductions in writhing correlated with reduced trafficking of myeloid cells into the peritoneal cavity. We show that CCR1 is highly expressed on circulating neutrophils and their depletion decreases acetic acid-induced writhing. However, administration of neutrophils into the peritoneal cavity did not enhance acetic acid-induced writhing in wild-type (WT) or CCR1-/- mice. Additionally, selective knockout of CCR1 in either the hematopoietic or non-hematopoietic compartments also reduced writhing. Together these data suggest that CCR1 functions to significantly modulate pain by controlling neutrophil trafficking to the inflammatory site and having an unexpected role on non-hematopoietic cells. As inflammatory diseases are often accompanied with infiltrating immune cells at the inflammatory site and pain, CCR1 antagonism may provide a dual benefit by restricting leukocyte trafficking and reducing pain.

Nonsteroidal dissociated glucocorticoid agonists containing azaindoles as steroid A-ring mimetics.

Syntheses and structure-activity relationships (SAR) of nonsteroidal glucocorticoid receptor (GR) agonists are described. These compounds contain azaindole moieties as A-ring mimetics and display various degrees of in vitro dissociation between gene transrepression and transactivation. Collagen induced arthritis studies in mouse have demonstrated that in vitro dissociated compounds (R)-16 and (R)-37 have steroid-like anti-inflammatory properties with improved metabolic side effect profiles, such as a reduced increase in body fat and serum insulin levels, compared to steroids.

Identification of dissociated non-steroidal glucocorticoid receptor agonists.

A new series of ligands for the glucocorticoid receptor (GR) is described. SAR development was guided by docking 3 into the GR active site and optimizing an unsubstituted phenyl ring for key interactions found in the steroid A-ring binding pocket. To identify compounds with an improved side effect profile over marketed steroids the functional activity of compounds was evaluated in cell based assays for transactivation (aromatase) and transrepression (IL-6). Through this effort, 36 has been identified as a partial agonist with a dissociated profile in these cell based assays.

Trifluoromethyl group as a pharmacophore: effect of replacing a CF3 group on binding and agonist activity of a glucocorticoid receptor ligand.

Compound 1, a potent glucocorticoid receptor ligand, contains a quaternary carbon bearing trifluoromethyl and hydroxyl groups. This paper describes the effect of replacing the trifluoromethyl group on binding and agonist activity of the GR ligand 1. The results illustrate that replacing the CF3 group with a cyclohexylmethyl or benzyl group maintains the GR binding potency. These substitutions alter the functional behavior of the GR ligands from agonists to antagonists. Docking studies suggest that the benzyl analog 19 binds in a similar fashion as the GR antagonist, RU486. The central benzyl group of 19 and the C-11 dimethylaniline moiety of RU486 overlay. Binding of compound 19 is believed to force helix 12 to adopt an open conformation and this leads to the antagonist properties of the non-CF3 ligands carrying a large group at the center of the molecule.