Novel amide and imidazole compounds as potent hematopoietic prostaglandin D2 synthase inhibitors.

In seeking novel and potent small molecule hematopoietic prostaglandin D2 synthase (H-PGDS) inhibitors as potential therapies for PGD2-mediated diseases and conditions, we explored a series comprising multiple aryl/heteroaryl rings attached in a linear arrangement. Each compound incorporates an amide or imidazole "linker" between the pyrimidine or pyridine "core" ring and the "tail" ring system. We synthesized and screened twenty analogs by fluorescence polarization binding assay, thermal shift assay, glutathione S-transferase inhibition assay, and a cell-based assay measuring suppression of LPS-induced PGD2 stimulation. Amide analogs show ten-fold greater shift in the thermal shift assay in the presence of glutathione (GSH) versus the same assay run in the absence of GSH. The imidazole analogs did not produce a significant change in thermal shift between the two assay conditions, suggesting a possible stabilization effect of the amide linker in the synthase-GSH-inhibitor complex. Imidazole analog 23, (KMN-010034) demonstrates superior potency across the in vitro assays and good in vitro metabolic stability in both human and guinea pig liver microsomes.

KMN-159, a novel EP4 receptor selective agonist, stimulates osteoblastic differentiation in cultured whole rat bone marrow.

KMN-159 is the lead compound from a series of novel difluorolactam prostanoid EP4 receptor agonists aimed at inducing local bone formation while avoiding the inherent side effects of systemic EP4 activation. KMN-159 is a potent, selective small molecule possessing pharmacokinetic properties amenable to local administration. Unfractionated rat bone marrow cells (BMCs) were treated once at plating with escalating doses of KMN-159 (1 pM to 10 µM). The resulting elevated alkaline phosphatase (ALP) levels measured 9 days post-dose are consistent with increased osteoblastic differentiation and exposure to KMN-159 at low nanomolar concentrations for as little as 30 min was sufficient to induce complete osteoblast differentiation of the BMCs from both sexes and regardless of age. ALP induction was blocked by an EP4 receptor antagonist but not by EP1 or EP2 receptor antagonists and was not induced by EP2 or EP3 receptor agonists. Addition of BMCs to plates coated with KMN-159 24 days earlier resulted in ALP activation, highlighting the chemical stability of the compound. The expression of phenotype markers such as ALP, type I collagen, and osteocalcin was significantly elevated throughout the osteoblastic differentiation timecourse initiated by KMN-159 stimulation. An increased number of tartrate-resistant acid phosphatase-positive cells was observed KMN-159 or PGE2 treated BMCs but only in the presence of exogenous receptor activator of nuclear factor kappa-Β ligand (RANKL). No change in the number of adipocytes was observed. KMN-159 also increased bone healing in a rat calvarial defect model with a healing rate equivalent to recombinant human bone morphogenetic protein-2. Our studies show that KMN-159 is able to stimulate osteoblastic differentiation with a very short time of exposure, supporting its potential as a therapeutic candidate for augmenting bone mass.

Improved homology modeling of the human & rat EP4 prostanoid receptors.

BACKGROUND: The EP4 prostanoid receptor is one of four GPCRs that mediate the diverse actions of prostaglandin E2 (PGE2). Novel selective EP4 receptor agonists would assist to further elucidate receptor sub-type function and promote development of therapeutics for bone healing, heart failure, and other receptor associated conditions. The rat EP4 (rEP4) receptor has been used as a surrogate for the human EP4 (hEP4) receptor in multiple SAR studies. To better understand the validity of this traditional approach, homology models were generated by threading for both receptors using the RaptorX server. These models were fit to an implicit membrane using the PPM server and OPM database with refinement of intra and extracellular loops by Prime (Schrödinger). To understand the interaction between the receptors and known agonists, induced-fit docking experiments were performed using Glide and Prime (Schrödinger), with both endogenous agonists and receptor sub-type selective, small-molecule agonists. The docking scores and observed interactions were compared with radioligand displacement experiments and receptor (rat & human) activation assays monitoring cAMP. RESULTS: Rank-ordering of in silico compound docking scores aligned well with in vitro activity assay EC50 and radioligand binding Ki. We observed variations between rat and human EP4 binding pockets that have implications in future small-molecule receptor-modulator design and SAR, specifically a S103G mutation within the rEP4 receptor. Additionally, these models helped identify key interactions between the EP4 receptor and ligands including PGE2 and several known sub-type selective agonists while serving as a marked improvement over the previously reported models. CONCLUSIONS: This work has generated a set of novel homology models of the rEP4 and hEP4 receptors. The homology models provide an improvement upon the previously reported model, largely due to improved solvation. The hEP4 docking scores correlates best with the cAMP activation data, where both data sets rank order Rivenprost>CAY10684 > PGE1 ≈ PGE2 > 11-deoxy-PGE1 ≈ 11-dexoy-PGE2 > 8-aza-11-deoxy-PGE1. This rank-ordering matches closely with the rEP4 receptor as well. Species-specific differences were noted for the weak agonists Sulprostone and Misoprostol, which appear to dock more readily within human receptor versus rat receptor.

Difluoromethylene at the γ-Lactam α-Position Improves 11-Deoxy-8-aza-PGE1 Series EP4 Receptor Binding and Activity: 11-Deoxy-10,10-difluoro-8-aza-PGE1 Analog (KMN-159) as a Potent EP4 Agonist.

A series of small-molecule full agonists of the prostaglandin E2 type 4 (EP4) receptor have been generated and evaluated for binding affinity and cellular potency. KMN-80 and its gem-difluoro analog KMN-159 possess high selectivity relative to other prostanoid receptors. Difluoro substitution is positioned alpha to the lactam ring carbonyl and results in KMN-159's fivefold increase in potency versus KMN-80. The two analogs exhibit electronic and conformational variations, including altered nitrogen hybridization and lactam ring puckering, that may drive the observed difluoro-associated increased potency within this four-compound series.

Discovery of a series of 2-(1H-pyrazol-1-yl)pyridines as ALK5 inhibitors with potential utility in the prevention of dermal scarring.

A series of 2-(1H-pyrazol-1-yl)pyridines are described as inhibitors of ALK5 (TGFß receptor I kinase). Modeling compounds in the ALK5 kinase domain enabled some optimization of potency via substitutions on the pyrazole core. One of these compounds PF-03671148 gave a dose dependent reduction in TGFß induced fibrotic gene expression in human fibroblasts. A similar reduction in fibrotic gene expression was observed when PF-03671148 was applied topically in a rat wound repair model. Thus these compounds have potential utility for the prevention of dermal scarring.

The discovery of the benzhydroxamate MEK inhibitors CI-1040 and PD 0325901.

A novel series of benzhydroxamate esters derived from their precursor anthranilic acids have been prepared and have been identified as potent MEK inhibitors. 2-(2-Chloro-4-iodo-phenylamino)-N-cyclopropylmethoxy-3,4-difluoro-benzamide, CI-1040, was the first MEK inhibitor to demonstrate in vivo activity in preclinical animal models and subsequently became the first MEK inhibitor to enter clinical trial. CI-1040 suffered however from poor exposure due to its poor solubility and rapid clearance, and as a result, development of the compound was terminated. Optimization of the diphenylamine core and modification of the hydroxamate side chain for cell potency, solubility, and exposure with oral delivery resulted in the discovery of the clinical candidate N-(2,3-dihydroxy-propoxy)-3,4-difluoro-2-(2-fluoro-4-iodo-phenylamino)-benzamide PD 0325901.

Central role of the MEK/ERK MAP kinase pathway in a mouse model of rheumatoid arthritis: potential proinflammatory mechanisms.

OBJECTIVE: To evaluate the role of the MEK/ERK MAP kinase pathway in murine collagen-induced arthritis (CIA) using the selective MEK inhibitor PD184352. We examined the effects of the inhibitor in cytokine-stimulated synovial fibroblasts and in cytokine-induced arthritis in rabbits to investigate its antiinflammatory mechanisms. METHODS: Murine CIA was used to assess the effects of the selective MEK inhibitor on paw edema, clinical scores, weight loss, histopathologic features, and joint levels of p-ERK. Western blotting and immunohistochemistry techniques were used to assess p-ERK in human and rabbit synovial fibroblasts and synovial tissue from rheumatoid arthritis (RA) patients. Interleukin-1alpha (IL-1alpha)-stimulated stromelysin production in rabbit synovial fibroblasts was assessed by enzyme-linked immunosorbent assay. A rabbit IL-1alpha-induced arthritis model was used to assess the effects of the inhibitor on IL-1alpha-induced MEK activity, stromelysin production, and cartilage degradation. RESULTS: In the CIA model, PD184352 inhibited paw edema and clinical arthritis scores in a dose-dependent manner. Disease-induced weight loss and histopathologic changes were also significantly improved by treatment. Inhibition of disease-induced p-ERK levels in the joints was seen with the inhibitor. Levels of p-ERK in the synovium were higher in RA patients than in normal individuals. PD184352 reduced IL-1alpha-induced p-ERK levels in human RA synovial fibroblasts. The production of p-ERK and stromelysin was also inhibited in IL-1alpha-stimulated rabbit synovial fibroblasts. We observed IL-1alpha-induced p-ERK in the synovial lining, subsynovial vasculature, and articular chondrocytes. IL-1alpha-induced stromelysin production and proteoglycan loss from the articular cartilage were reduced by PD184352. CONCLUSION: These data demonstrate the inhibition of murine CIA by PD184352, support the hypothesis that antiinflammatory activity contributes to the mechanism of action of the inhibitor, and suggest that a selective inhibitor may effectively treat RA and other inflammatory disorders.

4-anilino-5-carboxamido-2-pyridone derivatives as noncompetitive inhibitors of mitogen-activated protein kinase kinase.

A new series of MEK1 inhibitors, the 4-anilino-5-carboxamido-2-pyridones, were designed and synthesized using a combination of medicinal chemistry, computational chemistry, and structural elucidation. The effect of variation in the carboxamide side chain, substitution on the pyridone nitrogen, and replacement of the 4'-iodide were all investigated. This study afforded several compounds which were either equipotent or more potent than the clinical candidate CI-1040 (1) in an isolated enzyme assay, as well as murine colon carcinoma (C26) cells, as measured by suppression of phosphorylated ERK substrate. Most notably, pyridone 27 was found to be more potent than 1 in vitro and produced a 100% response rate at a lower dose than 1, when tested for in vivo efficacy in animals bearing C26 tumors.