Development of Cilofexor, an intestinally-biased Farnesoid X Receptor agonist, for the treatment of fatty liver disease.

The farnesoid X receptor (FXR) is a nuclear receptor that controls bile acid, lipid, and cholesterol metabolism. FXR-targeted drugs have shown promise in late-stage clinical trials for non-alcoholic steatohepatitis. Herein, we used clinical results from our first non-steroidal FXR agonist, Px-102 (4-[2-[2-chloro-4-[[5-cyclopropyl-3-(2,6-dichlorophenyl)-4-isoxazolyl]methoxy]phenyl]cyclopropyl] benzoic acid), to develop cilofexor, a potent, non-steroidal FXR agonist with a more manageable safety profile. Px-102 demonstrated the anticipated pharmacodynamic (PD) effects in healthy volunteers but caused a 2-fold increase in alanine aminotransferase (ALT) activity and changes in cholesterol levels. These data guided development of a high fat diet mouse model to screen FXR agonists based on ALT and cholesterol changes. Cilofexor was identified to elicit only minor changes in these parameters. The differing effects of cilofexor and Px-102 on ALT/cholesterol in the model could not be explained by potency or specificity, and we hypothesized that the relative contribution of intestinal and liver FXR activation may be responsible. Gene expression analysis from rodent studies revealed that cilofexor, but not Px-102, had a bias for FXR transcriptional activity in the intestine compared to the liver. Fluorescent imaging in hepatoma cells demonstrated similar subcellular localization for cilofexor and Px-102, but cilofexor was more rapidly washed out, consistent with a lower membrane residence time contributing to reduced hepatic transcriptional effects. Cilofexor demonstrated antisteatotic and antifibrotic efficacy in rodent models and antisteatotic efficacy in a monkey model, with the anticipated PD and a manageable safety profile in human phase I studies. Significance Statement FXR (farnesoid X receptor) agonists have shown promise in treating non-alcoholic steatohepatitis and other liver diseases in the clinic, but balancing efficacy with undesired side effects has been difficult. Here, we examined the preclinical and clinical effects of the first-generation FXR agonist, Px-102 (4-[2-[2-chloro-4-[[5-cyclopropyl-3-(2,6-dichlorophenyl)-4-isoxazolyl]methoxy]phenyl]cyclopropyl] benzoic acid), to enable the selection of an analog, cilofexor, with unique properties that reduced side effects yet maintained efficacy. Cilofexor is one of few remaining FXR agonists in clinical development.

Discovery and optimization of substituted oxalamides as novel heme-displacing IDO1 inhibitors.

Since the advent of antibody checkpoint inhibitors as highly efficient drugs for cancer treatment, the development of immunomodulating small molecules in oncology has gained great attention. Drug candidates targeting IDO1, a key enzyme in tryptophan metabolism, are currently under clinical investigation in combination with PD-1/PD-L1 agents as well as with other established anti-tumor therapeutics. A ligand based design approach from hydroxyamidine 4 that aimed at heme-binding IDO1 inhibitors resulted in new compounds with moderate IDO1 potency. A hybrid structure design that made use of the linrodostat structure (2) led to oxalamide derived, heme-displacing IDO1 inhibitors with high cell-based IDO1 potency and a favorable ADME/PK profile.

Discovery of highly potent heme-displacing IDO1 inhibitors based on a spirofused bicyclic scaffold.

Through structural modification of an oxalamide derived chemotype, a novel class of highly potent, orally bioavailable IDO1-specific inhibitors was identified. Representative compound 18 inhibited human IDO1 with IC50 values of 3.9 nM and 52 nM in a cellular and human whole blood assay, respectively. In vitro assessment of the ADME properties of 18 demonstrated very high metabolic stability. Pharmacokinetic profiling in mice showed a significantly reduced clearance compared to the oxalamides. In a mouse pharmacodynamic model 18 nearly completely suppressed lipopolysaccharide-induced kynurenine production. Hepatocyte data of 18 suggest the human clearance to be in a similar range to linrodostat (1).

Discovery and optimization of new oxadiazole substituted thiazole RORγt inverse agonists through a bioisosteric amide replacement approach.

Starting from previously identified thiazole-2-carboxamides exemplified by compound 1/6, two new series of RORγt inverse agonists with significantly improved aqueous solubility, ADME parameters and oral PK properties were discovered. These scaffolds were identified from a bioisosteric amide replacement approach. Amongst the variety of heterocycles explored, a 1,3,4-oxadiazole led to compounds with the best overall profile for SAR development and in vivo exploration. In an ex vivo mouse PD model, concentration dependent efficacy was demonstrated and compounds 3/5 and 6/3 were profiled in a 5-day rat tolerability study.

Optimization and biological evaluation of thiazole-bis-amide inverse agonists of RORγt.

The nuclear receptor retinoic acid receptor-related orphan receptor gamma t (RORγt) is a transcription factor that drives Th17 cell differentiation and IL-17 production in both innate and adaptive immune cells. The IL-23/IL-17 pathway is implicated in major autoimmune and inflammatory diseases. RORγt lies at the core of this pathway and represents an attractive opportunity for intervention with small molecule therapeutics. Despite diverse chemical series having been reported, combining high potency and nuclear receptor selectivity with good physicochemical properties remains a challenging endeavor in the field of RORγt drug discovery. We recently described the discovery and evaluation of a new class of potent and selective RORγt inverse agonists based on a thiazole scaffold. Herein we describe the successful optimization of this class by incorporation of an additional amide moiety at the 4-position of the thiazole core. In several optimization cycles, we have reduced human PXR activation, improved solubility, and increased potency while maintaining nuclear receptor selectivity. X-ray crystallographic analysis of compound 1g bound in the sterol binding site of the ligand binding domain of RORγt was largely consistent with an earlier structure, guiding further insight into the molecular mechanism for RORγt inhibition with this series. Compound 1g is orally bioavailable, potent in a human whole blood assay and proved to be efficacious in an ex-vivo IL-17A assay, and was selected for preclinical evaluation.

Discovery of Hydroxyamidine Based Inhibitors of IDO1 for Cancer Immunotherapy with Reduced Potential for Glucuronidation.

Following the impressive success of checkpoint inhibitors in the treatment of cancer, combinations of IDO1 inhibitors with PD-1/PD-L1 antibodies are in clinical development aiming to increase response rates. Using the hydroxyamidine pharmacophore of the IDO1 inhibitor INCB14943 as a starting point for the design of new inhibitors, the potential shortcomings of extensive hydroxyamidine glucuronidation in humans was addressed. Compounds were optimized using a stability assay with recombinant UGT1A9 enzyme together with the measurement of glucuronide formation in human hepatocytes. Optimized analog 24 showed cellular and biochemical IDO1 IC50 values in the low nanomolar range, a suitable in vitro ADME/PK profile, and efficacy in an animal model of cancer. In a humanized liver mouse model the lead compound exhibited significantly reduced glucuronidation compared to epacadostat (2).

Identification and biological evaluation of thiazole-based inverse agonists of RORγt.

The nuclear receptor retinoic acid receptor-related orphan receptor gamma t (RORγt) is a transcription factor that drives Th17 cell differentiation and IL-17 production in both innate and adaptive immune cells. The IL-23/IL-17 pathway is implicated in major autoimmune and inflammatory diseases. RORγt lies at the core of this pathway and represents an attractive opportunity for intervention with a small molecule. Despite diverse chemical series having been reported, combining high potency and nuclear receptor selectivity with good physicochemical properties remains a challenging endeavor in the field of RORγt drug discovery. We describe the discovery and evaluation of a new class of potent and selective RORγt inverse agonists based on a thiazole core. Acid analog 1j demonstrated oral bioavailability in rats and was potent in a human whole blood assay, suggesting potential utility in treating autoimmune and inflammatory diseases such as psoriasis. X-ray crystallographic data helped to elucidate the molecular mechanism for RORγt inhibition with this series.

Novel substituted isoxazole FXR agonists with cyclopropyl, hydroxycyclobutyl and hydroxyazetidinyl linkers: Understanding and improving key determinants of pharmacological properties.

Several isoxazole-containing series of FXR agonists have been published over the last 15years, subsequent to the prototypical amphiphilic 'hammerhead'-type structure that was originally laid out by GW4064, the first potent synthetic FXR agonist. A set of novel compounds where the hammerhead is connected to the terminal carboxylic acid-bearing aryl or heteroaryl moiety by either a cyclopropyl, a hydroxycyclobutyl or a hydroxyazetidinyl linker was synthesized in order to improve upon the ADME properties of such isoxazoles. The resulting compounds all demonstrated high potencies at the target receptor FXR but with considerable differences in their physicochemical and in vivo profiles. The structure-activity relationships for key chemical features that have a major impact on the in vivo pharmacology of this series are discussed.

Knocking on FXR's door: the "hammerhead"-structure series of FXR agonists - amphiphilic isoxazoles with potent in vitro and in vivo activities.

The Farnesoid X Receptor (FXR) was recently validated in clinical studies using the bile acid analogue Obeticholic Acid (OCA) as an attractive drug target for liver diseases such as Primary Biliary Cirrhosis (PBC) or Non-alcoholic Steatohepatitis (NASH). OCA, however, turned out to induce cholesterol- related side effects upon prolonged treatment and it shows bile acid like pharmacokinetics. The quest for synthetic non-steroidal FXR agonists with general drug likeliness and improved pharmacokinetic and - dynamic properties has started more than a decade ago: The first non-steroidal and selective FXR agonist with decent submicromolar potency, GW4064, was patented in 1998 and published in 2000. Since then, many pharmaceutical companies have taken GW4064 as a structural template for their efforts in identifying novel patentable FXR agonists with the GW-derived trisubstituted isoxazole general structure. However, so far only one compound out of these different series has made it into the early stages of clinical development: The Px-102/Px-104 from Phenex is currently tested in a phase IIa study in patients with Non-Alcoholic Fatty Liver Disease (NAFLD). In this review we try to summarize from the patent and scientific literature the attempts to improve the GW4064 structure into different directions. Furthermore, we suggest directions for further improvements of this special class of synthetic FXR agonists which all display the typical "hammerhead"-conformation in the FXR ligand binding pocket that provides the basis for their impressive in vitro and in vivo potencies.

Discovery and evaluation of a non-Zn chelating, selective matrix metalloproteinase 13 (MMP-13) inhibitor for potential intra-articular treatment of osteoarthritis.

Osteoarthritis (OA) is a nonsystemic disease for which no oral or parenteral disease-modifying osteoarthritic drug (DMOAD) is currently available. Matrix metalloproteinase 13 (MMP-13) has attracted attention as a target with disease-modifying potential because of its major role in tissue destruction associated with OA. Being localized to one or a few joints, OA is amenable to intra-articular (IA) therapy, which has distinct advantages over oral therapies in terms of increasing therapeutic index, by maximizing drug delivery to cartilage and minimizing systemic exposure. Here we report on the synthesis and biological evaluation of a non-zinc binding MMP-13 selective inhibitor, 4-methyl-1-(S)-({5-[(3-oxo-3,4-dihydro-2H-benzo[1,4]oxazin-6-ylmethyl)carbamoyl]pyrazolo[1,5-a]pyrimidine-7-carbonyl}amino)indan-5-carboxylic acid (1), that is uniquely suited as a potential IA-DMOAD: it has long durability in the joint, penetrates cartilage effectively, exhibits nearly no detectable systemic exposure, and has remarkable efficacy.

Synthesis and pharmacological validation of a novel series of non-steroidal FXR agonists.

To overcome the known liabilities of GW4064 a series of analogs were synthesized where the stilbene double bond is replaced by an oxymethylene or amino-methylene linker connecting a terminal benzoic acid with a substituted heteroaryl in the middle ring position. As a result we discovered compounds with increased potency in vitro that cause dose-dependent reduction of plasma triglycerides and cholesterol in db/db mice down to 2 x 1 mg/kg/day upon oral administration.

A new class of potent matrix metalloproteinase 13 inhibitors for potential treatment of osteoarthritis: Evidence of histologic and clinical efficacy without musculoskeletal toxicity in rat models.

OBJECTIVE: Matrix metalloproteinases (MMPs) have long been considered excellent targets for osteoarthritis (OA) treatment. However, clinical utility of broad-spectrum MMP inhibitors developed for this purpose has been restricted by dose-limiting musculoskeletal side effects observed in humans. This study was undertaken to identify a new class of potent and selective MMP-13 inhibitors that would provide histologic and clinical efficacy without musculoskeletal toxicity. METHODS: Selectivity assays were developed using catalytic domains of human MMPs. Freshly isolated bovine articular cartilage or human OA cartilage was used in in vitro cartilage degradation assays. The rat model of monoiodoacetate (MIA)-induced OA was implemented for assessing the effects of MMP-13 inhibitors on cartilage degradation and joint pain. The surgical medial meniscus tear model in rats was used to evaluate the chondroprotective ability of MMP-13 inhibitors in a chronic disease model of OA. The rat model of musculoskeletal side effects (MSS) was used to assess whether selective MMP-13 inhibitors have the joint toxicity associated with broad-spectrum MMP inhibitors. RESULTS: A number of non-hydroxamic acid-containing compounds that showed a high degree of potency for MMP-13 and selectivity against other MMPs were designed and synthesized. Steady-state kinetics experiments and Lineweaver-Burk plot analysis of rate versus substrate concentration with one such compound, ALS 1-0635, indicated linear, noncompetitive inhibition, and Dixon plot analysis from competition studies with a zinc chelator (acetoxyhydroxamic acid) and ALS 1-0635 demonstrated nonexclusive binding. ALS 1-0635 inhibited bovine articular cartilage degradation in a dose-dependent manner (48.7% and 87.1% at 500 nM and 5,000 nM, respectively) and was effective in inhibiting interleukin-1alpha- and oncostatin M-induced C1,C2 release in human OA cartilage cultures. ALS 1-0635 modulated cartilage damage in the rat MIA model (mean +/- SEM damage score 1.3 +/- 0.3, versus 2.2 +/- 0.4 in vehicle-treated animals). Most significantly, when treated twice daily with oral ALS 1-0635, rats with surgically induced medial meniscus tear exhibited histologic evidence of chondroprotection and reduced cartilage degeneration, without observable musculoskeletal toxicity. CONCLUSION: The compounds investigated in this study represent a novel class of MMP-13 inhibitors. They are mechanistically distinct from previously reported broad-spectrum MMP inhibitors and do not exhibit the problems previously associated with these inhibitors, including selectivity, poor pharmacokinetics, and MSS liability. MMP-13 inhibitors exert chondroprotective effects and can potentially modulate joint pain, and are, therefore, uniquely suited as potential disease-modifying osteoarthritis drugs.