Discovery and lead optimisation of a potent,selective and orally bioavailable RARβ agonist for the potential treatment of nerve injury

Oxadiazole replacement of an amide linkage in an RARα agonist template 1, followed by lead optimisation, has produced a highly potent and selective RARβ agonist 4-(5-(4,7-dimethylbenzofuran-2-yl)-1,2,4-oxadiazol-3-yl)benzoic acid (10) with good oral bioavailability in the rat and dog. This molecule increases neurite outgrowth in vitro and induces sensory axon regrowth in vivo in a rodent model of avulsion and crush injury, and thus has the potential for the treatment of nerve injury.     

Synthesis and evaluation of novel α-heteroaryl-phenylpropanoic acid derivatives as PPARα/γ dual agonists

The synthesis of a new series of phenylpropanoic acid derivatives incorporating an heteroaryl group at the α-position and their evaluation for binding and activation of PPARα and PPARγ are presented in this report. Among the new compounds, (S)-3-{4-[3-(5-methyl-2-phenyl-oxazol-4-yl)-propyl]-phenyl}-2-1,2,3-triazol-2-yl-propionic acid (17j), was identified as a potent human PPARα/γ dual agonist (EC₅₀ = 0.013 and 0.061 μM, respectively) with demonstrated oral bioavailability in rat and dog. 17j was shown to decrease insulin levels, plasma glucose, and triglycerides in the ZDF female rat model. In the human apolipoprotein A-1/CETP transgenic mouse model 17j produced increases in hApoA1 and HDL-C and decreases in plasma triglycerides. The increased potency for binding and activation of both PPAR subtypes observed with 17j when compared to previous analogs in this series was explained based on results derived from crystallographic and modeling studies.     

The potential signaling pathway between peroxisome proliferator-activated receptor gamma and retinoic acid receptor alpha in renal interstitial fibrosis disease

Abstract

Peroxisome proliferator-activated receptorγ (PPARγ) can regulate the process of cell apoptosis and is related to the progression of renal disorders. Retinoic acid receptor alpha (RARα) is one of the nuclear receptors involved in a variety of kidney diseases. Renal interstitial fibrosis (RIF) is a common denominator of chronic kidney disease (CKD). This study investigated whether a potential signaling pathway existed between PPARγ and RARα in RIF rats with unilateral ureteral obstruction (UUO). The rats were randomly divided into four groups: a model group subjected to UUO (GU), and three other groups treated with rosiglitazone sodium (GRS), GW9662 and dimethyl sulfoxide (DMSO), n?=?40, respectively. Renal tissues were collected two and four weeks after post-surgery. The relevant indicators were detected. In comparison with the GU group, the expressions of PPARγ and RARα (protein and mRNA) were increased in the GRS group, and decreased in the GW9662 group (all p?<?0.01). The RIF index, mRNA and protein expression of transforming growth factor-β1 (TGF-β1), and the protein expressions of collagen-IV (Col-IV) and fibronectin (FN) in the GRS group were more markedly reduced than those in the GU group; their levels in the GW9662 group were elevated (all p?<?0.01). PPARγ or RARα was negatively correlated to the RIF index, TGF-β1, Col-IV and FN. PPARγ was positively correlated with RARα (all p?<?0.01). In conclusion, PPARγ agonist can elevate the expression of PPARγ or RARα in RIF rats. There might be a potential signaling pathway between PPARγ and RARα in RIF disease.     

Synthesis and structure–activity relationships of 2-aryl-4-oxazolylmethoxy benzylglycines and 2-aryl-4-thiazolylmethoxy benzylglycines as novel,potent PPARα selective activators- PPARα and PPARγ selectivity modulation

The synthesis and follow-up SAR studies of our development candidate 1 by incorporating 2-aryl-4-oxazolylmethoxy and 2-aryl-4-thiazolylmethoxy moieties into the oxybenzylglycine framework of the PPARα/γ dual agonist muraglitazar is described. SAR studies indicate that different substituents on the aryloxazole/thiazole moieties as well as the choice of carbamate substituent on the glycine moiety can significantly modulate the selectivity of PPARα versus PPARγ. Potent, highly selective PPARα activators 2a and 2l, as well as PPARα activators with significant PPARγ activity, such as 2s, were identified. The in vivo pharmacology of these compounds in preclinical animal models as well as their ADME profiles are discussed.     

RARα and RARγ reciprocally control K5+ progenitor cell expansion in developing salivary glands

Understanding the mechanisms of controlled expansion and differentiation of basal progenitor cell populations during organogenesis is essential for developing targeted regenerative therapies. Since the cytokeratin 5-positive (K5⁺) basal epithelial cell population in the salivary gland is regulated by retinoic acid signaling, we interrogated how isoform-specific retinoic acid receptor (RAR) signaling impacts the K5⁺ cell population during salivary gland organogenesis to identify RAR isoform-specific mechanisms that could be exploited in future regenerative therapies. In this study, we utilized RAR isoform-specific inhibitors and agonists with murine submandibular salivary gland organ explants. We determined that RARα and RARγ have opposing effects on K5⁺ cell cycle progression and cell distribution. RARα negatively regulates K5⁺ cells in both whole organ explants and in isolated epithelial rudiments. In contrast, RARγ is necessary but not sufficient to positively maintain K5⁺ cells, as agonism of RARγ alone failed to significantly expand the population. Although retinoids are known to stimulate differentiation, K5 levels were not inversely correlated with differentiated ductal cytokeratins. Instead, RARα agonism and RARγ inhibition, corresponding with reduced K5, resulted in premature lumenization, as marked by prominin-1. With lineage tracing, we demonstrated that K5⁺ cells have the capacity to become prominin-1⁺ cells. We conclude that RARα and RARγ reciprocally control K5⁺ progenitor cells endogenously in the developing submandibular salivary epithelium, in a cell cycle-dependent manner, controlling lumenization independently of keratinizing differentiation. Based on these data, isoform-specific targeting RARα may be more effective than pan-RAR inhibitors for regenerative therapies that seek to expand the K5⁺ progenitor cell pool. Summary statement: RARα and RARγ reciprocally control K5⁺ progenitor cell proliferation and distribution in the developing submandibular salivary epithelium in a cell cycle-dependent manner while regulating lumenization independently of keratinizing differentiation.     

Novel sulfamoyl benzamides as selective CB2 agonists with improved in vitro metabolic stability

A lead optimization campaign in our previously reported sulfamoyl benzamide class of CB₂ agonists was conducted to improve the in vitro metabolic stability profile in this series while retaining high potency and selectivity for the CB₂ receptor. From this study, compound 14, N-(3,4-dimethyl-5-(morpholinosulfonyl)phenyl)-2,2-dimethylbutanamide, was identified as a potent and selective CB₂ agonist exhibiting moderate in vitro metabolic stability and oral bioavailability. Compound 14 demonstrated in vivo efficacy in a rat model of post-surgical pain.     

Retinoic acid receptor signalling directly regulates osteoblast and adipocyte differentiation from mesenchymal progenitor cells

Low and high serum retinol levels are associated with increased fracture risk and poor bone health. We recently showed retinoic acid receptors (RARs) are negative regulators of osteoclastogenesis. Here we show RARs are also negative regulators of osteoblast and adipocyte differentiation. The pan-RAR agonist, all-trans retinoic acid (ATRA), directly inhibited differentiation and mineralisation of early osteoprogenitors and impaired the differentiation of more mature osteoblast populations. In contrast, the pan-RAR antagonist, IRX4310, accelerated differentiation of early osteoprogenitors. These effects predominantly occurred via RARγ and were further enhanced by an RARα agonist or antagonist, respectively. RAR agonists similarly impaired adipogenesis in osteogenic cultures. RAR agonist treatment resulted in significant upregulation of the Wnt antagonist, Sfrp4. This accompanied reduced nuclear and cytosolic β-catenin protein and reduced expression of the Wnt target gene Axin2, suggesting impaired Wnt/β-catenin signalling. To determine the effect of RAR inhibition in post-natal mice, IRX4310 was administered to male mice for 10 days and bones were assessed by µCT. No change to trabecular bone volume was observed, however, radial bone growth was impaired. These studies show RARs directly influence osteoblast and adipocyte formation from mesenchymal cells, and inhibition of RAR signalling in vivo impairs radial bone growth in post-natal mice.     

Recent advances in the design of RAR α and RAR β agonists as orally bioavailable drugs. A review

Retinoic acid receptors (RARs) α, β, and γ are members of the nuclear receptor superfamily. Compounds which bind to and activate the RARs are termed retinoids which regulate a wide variety of biological processes such as vertebrate embryonic morphogenesis and organogenesis, cell growth arrest, differentiation, and apoptosis, as well as their disorders. Although many synthetic selective RARα, RARβ, and RARγ agonists have been designed and prepared, these have generally been lipophilic acids without good drug-like properties and with low oral bioavailability. Recently this has been changing and drug design approaches to highly potent and selective RARα and RARβ agonists with low lipophilicity that are orally bioavailable and less toxic have been developed, that have a range of potential therapeutic uses. This review covers these new advances.     

Molecular modelling,synthesis, and biological evaluations of a 3,5-disubstituted isoxazole fatty acid analogue as a PPARα-selective agonist

The peroxisome proliferator activated receptors (PPARs) are important drug targets in treatment of metabolic and inflammatory disorders. Fibrates, acting as PPARα agonists, have been widely used lipid-lowering agents for decades. However, the currently available PPARα targeting agents show low subtype-specificity and consequently a search for more potent agonists have emerged. In this study, previously isolated oxohexadecenoic acids from the marine algae Chaetoceros karianus were used to design a PPARα-specific analogue. Herein we report the design, synthesis, molecular modelling studies and biological evaluations of the novel 3,5-disubstituted isoxazole analogue 6-(5-heptyl-1,2-oxazol-3-yl)hexanoic acid (1), named ADAM. ADAM shows a clear receptor preference and significant dose-dependent activation of PPARα (EC₅₀ = 47 µM) through its ligand-binding domain (LBD). Moreover, ADAM induces expression of important PPARα target genes, such as CPT1A, in the Huh7 cell line and primary mouse hepatocytes. In addition, ADAM exhibits a moderate ability to regulate PPARγ target genes and drive adipogenesis. Molecular modelling studies indicated that ADAM docks its carboxyl group into opposite ends of the PPARα and -γ LBD. ADAM interacts with the receptor-activating polar network of amino acids (Tyr501, His447 and Ser317) in PPARα, but not in PPARγ LBD. This may explain the lack of PPARγ agonism, and argues for a PPARα-dependent adipogenic function. Such compounds are of interest towards developing new lipid-lowering remedies.     

Discovery of N-(5,6-diarylpyridin-2-yl)amide derivatives as potent and selective A2B adenosine receptor antagonists

The synthesis and SAR of a series of N-(5,6-diarylpyridin-2-yl)amide derivatives as potent A_2B adenosine receptor antagonists is described. Several compounds showed good selectivity versus other adenosine receptors. The potent and selective analogue 9 was shown to have good oral bioavailability in the rat.     

Evaluation of 1,2,5-thiadiazoles as modulators of M1/M5 muscarinic receptor subtypes

Studies have demonstrated the presence of allosteric binding sites on each of the muscarinic acetylcholine receptor (mAChR) subtypes. Since most drugs targeting muscarinic receptors bind to the highly conserved orthosteric binding site, they fail to achieve appreciable subtype selectivity. Targeting non-conserved allosteric sites may provide a new way of enhancing selectivity for individual subtypes of muscarinic receptor. Tetra(ethyleneglycol)(3-methoxy-1,2,5-thiadiazol-4-yl)[3-(1-methyl-1,2,5,6-tetrahydropyrid-3-yl)-1,2,5-thiadiazol-4-yl] ether, CDD-0304 (10), was found to be a M_1/2/4 selective muscarinic agonist and might prove useful in treating the symptoms associated with schizophrenia (J. Med. Chem. 2003, 46, 4273). It was hypothesized that the observed subtype selectivity demonstrated by 10 may be due to its ability to function as a bitopic ligand (J. Med. Chem. 2006, 49, 7518). To further investigate this possibility, a novel series of compounds was synthesized using a 1,2,5-thiadiazole moiety along with varying lengths of a polyethylene glycol linker and terminal groups, for evaluation as potential allosteric modulators of muscarinic receptors. Preliminary biological studies were performed using carbachol to stimulate M₁ and M₅ receptors. No significant agonist activity was observed at either M₁ or M₅ receptors for any of the compounds. Compound 18, 2-(4-methoxy-1,2,5-thiadiazol-3-yloxy)-N,N-dimethylethanamine fumarate (CDD-0361F) was found to block the effects of carbachol at M₅ muscarinic receptors.     

Synthesis and evaluation of [(1R)-1-amino-2-(2,5-difluorophenyl)ethyl]cyclohexanes and 4-[(1R)-1-amino-2-(2,5-difluorophenyl)ethyl]piperidines as DPP-4 inhibitors

A series of 4-amino cyclohexanes and 4-substituted piperidines were prepared and evaluated for inhibition of DPP-4. Analog 20q displayed both good DPP-4 potency and selectivity against other proteases, while derivative 20k displayed long half life and modest oral bioavailability in rat. The most potent analog, 3-(5-aminocarbonylpyridyl piperidine 53j, displayed excellent DPP-4 activity with good selectivity versus other proline enzymes.     

Retinoic acid receptors are required for skeletal growth, matrix homeostasis and growth plate function in postnatal mouse

The retinoic acid receptors α, β and γ (RARα, RARβ and RARγ) are nuclear hormone receptors that regulate fundamental processes during embryogenesis, but their roles in skeletal development and growth remain unclear. To study skeletal-specific RAR function, we created conditional mouse mutants deficient in RAR expression in cartilage. We find that mice deficient in RARα and RARγ (or RARβ and RARγ) exhibit severe growth retardation obvious by about 3 weeks postnatally. Their growth plates are defective and, importantly, display a major drop in aggrecan expression and content. Mice deficient in RARα and RARβ, however, are virtually normal, suggesting that RARγ is essential. In good correlation, we find that RARγ is the most strongly expressed RAR in mouse growth plate and its expression characterizes the proliferative and pre-hypertrophic zones where aggrecan is strongly expressed also. By being avascular, those zones lack endogenous retinoids as indicated by previous RARE reporter mice and our direct biochemical measurements and thus, RARγ is likely to exert ligand-less repressor function. Indeed, our data indicate that: aggrecan production is enhanced by RARγ over-expression in chondrocytes under retinoid-free culture conditions; production is further boosted by co-repressor Zac1 or pharmacologic agents that enhance RAR repressor function; and RAR/Zac1 function on aggrecan expression may involve Sox proteins. In sum, our data reveal that RARs, and RARγ in particular, exert previously unappreciated roles in growth plate function and skeletal growth and regulate aggrecan expression and content. Since aggrecan is critical for growth plate function, its deficiency in RAR-mutant mice is likely to have contributed directly to their growth retardation.     

Detection of variable levels of RAR�� and RAR�� proteins in pluripotent and differentiating mouse embryonal carcinoma and mouse embryonic stem cells

Pluripotent mouse embryonal carcinoma (mEC) and mouse embryonic stem (mES) cells differentiate into several cell lineages upon retinoic acid (RA) addition. Differentiation is facilitated, in part, by RA activation of nuclear RA receptors (RARs) that bind to DNA response elements located in the promoters of target genes. The purpose of the studies reported here was to immunolocalize RARα and RARγ protein in mEC and mES cells and in their RA-induced differentiated progeny. Fixed cells were reacted with three different RARα antibodies and one RARγ antibody. Pluripotent and differentiated mEC and mES cells showed positive nuclear immunoreactivity with all antibodies tested. Two RARα antibodies also showed positive reactivity in the cytoplasm. Surprisingly, our results revealed variability in immunofluorescence intensity and in RARα and RARγ distribution from one cell to the other, suggesting that RARα and RARγ protein levels were not synchronous throughout the cell population. The results indicate that RARα and RARγ are present in pluripotent and differentiating mEC and mES cells and suggest that the expression of these proteins is dynamic.     

Piperazinyl-glutamate-pyridines as potent orally bioavailable P2Y12 antagonists for inhibition of platelet aggregation

Polymer-assisted solution-phase (PASP) parallel library synthesis was used to discover a piperazinyl-glutamate-pyridine as a P2Y₁₂ antagonist. Exploitation of this lead provided compounds with excellent inhibition of platelet aggregation as measured in a human platelet rich plasma (PRP) assay. Pharmacokinetic and physiochemical properties were optimized leading to compound (4S)-4-[({4-[4-(methoxymethyl)piperidin-1-yl]-6-phenylpyridin-2-yl}carbonyl)amino]-5-oxo-5-{4-[(pentyloxy)carbonyl]piperazin-1-yl}pentanoic acid 22J with good human PRP potency, selectivity, in vivo efficacy and oral bioavailability.