Patented Farnesoid X receptor modulators: a review (2019 - present).

INTRODUCTION: The Farnesoid X receptor (FXR) is a key transcription factor that is involved in the bile acid signaling network. The modulation of the FXR activity influences glucose and lipid homeostasis, reduces obesity and insulin resistance, as well as it regulates the pathogenesis of inflammatory and metabolic disorders. FXR ligands have therefore emerged in drug discovery as promising therapeutic agents for the prevention and treatment of gastrointestinal and liver diseases, including cancer. AREAS COVERED: Recent advances in the field of FXR modulators are reviewed, with a particular attention on patent applications filed in the past 5 years related to both the discovery and development of FXR targeting drugs. EXPERT OPINION: FXR agonists have proven their efficacy and safety in humans and have shown a significant potential as clinical agents to treat metabolic and inflammatory associated conditions. However, several challenges, including adverse events such as pruritus, remain to be solved. Current studies aim to gain insights into the pathophysiological mechanisms by which FXR regulates metabolism and inflammation in terms of tissue/organ/isoform-specificity, post-translational modifications and coregulatory proteins, on the route of novel, improved FXR modulators.

Multi-Gram Scale Synthesis and Characterization of Mometasone Furoate EP Impurity C.

Mometasone furoate is a synthetic corticosteroid used in the treatment of skin inflammatory conditions, hay fever and asthma. The industrial manufacturing routes to mometasone furoate are generally accompanied by the formation of numerous process impurities that need to be detected and quantified, as requested by regulatory authorities. The ready availability of such impurities in the required quantity and purity is therefore essential for toxicological studies, analytical method development and process validation. Herein, we report the multi-gram scale preparation of 21'-chloro-(16'α-methyl-3',11',20'-trioxo-pregna-1',4'-dien-17'-yl)-furan-2-carboxylate (mometasone furoate EP impurity C), one of the known impurities of mometasone furoate. This study also includes the systematic investigation of the final acylation step, as well as the characterization of the difuroate enol ether intermediate and its conversion to the target impurity C.

Chemical exploration of TGR5 functional hot-spots: Synthesis and structure-activity relationships of C7- and C23-Substituted cholic acid derivatives.

The activation of TGR5 bestows on bile acids the ability to modulate nongenomic signaling pathways, which are responsible of physiological actions including immunosuppressive and anti-inflammatory properties as well as the regulation of glucose metabolism and energy homeostasis. TGR5 agonists have therefore emerged in drug discovery and preclinical appraisals as promising compounds for the treatment of liver diseases and metabolic syndrome. In this study, we have been devising site-selected chemical modifications of the bile acid scaffold to provide novel chemical tools able to modulate the functions of TGR5 in different tissues. Biological results of the tested collection of semisynthetic cholic acid derivatives were used to extend the structure-activity relationships of TGR5 agonists and to clarify the molecular basis and functional role of TGR5 hot-spots in the receptor activation and selectivity. Some unexpected properties deriving from the molecular structure of bile acids have been unveiled as relevant to the receptor activation and may hence be used to design novel, selective and potent TGR5 agonists.

Turning a Tumor Microenvironment Pitfall into Opportunity: Discovery of Benzamidoxime as PD-L1 Ligand with pH-Dependent Potency.

PD-1/PD-L1 protein complex is attracting a great deal of interest as a drug target for the design of immune therapies able to block its assembly. Although some biologic drugs have entered clinical use, their poor response rate in patients are demanding further efforts to design small molecule inhibitors of PD-1/PD-L1 complex with higher efficacy and optimal physicochemical properties. Dysregulation of pH in the tumor microenvironment is indeed one of the key mechanisms promoting drug resistance and lack of response in cancer therapy. Integrating computational and biophysical approaches, herein we report a screening campaign that has led to identifying VIS310 as a novel ligand of PD-L1, with physicochemical properties enabling a pH-dependent binding potency. Additional optimization efforts by analogue-based screening have been instrumental to disclosing VIS1201, which exhibits improved binding potency against PD-L1 and is able to inhibit PD-1/PD-L1 complex formation in a ligand binding displacement assay. While providing preliminary structure-activity relationships (SARs) of a novel class of PD-L1 ligands, our results lay the foundation for the discovery of immunoregulatory small molecules resilient to tumor microenvironmental conditions for escaping drug-resistance mechanisms.

Domino synthesis of 5-aminoimidazoles from Strecker multicomponent adducts via ytterbium-promoted isocyanide insertion/5-exo-dig cyclization.

A new Lewis acid promoted domino isocyanide insertion/5-exo-dig cyclization of readily available Strecker 3-component adducts to 4-substituted 5-aminoimidazole derivatives is herein reported. Despite their potential as relevant heterocyclic scaffolds in medicinal chemistry programs, this class of compounds is still underrepresented, with current synthetic strategies poorly efficient in terms of timing and yields. To this end, we show how the exploitation of unconventional reactivities of isocyanides, promoted by ytterbium-triflate, could represent a key resource to enable a fast and easy access to such an unexplored area of the chemical space.

Development of 3α,7α-dihydroxy-6α-ethyl-24-nor-5ß-cholan-23-sulfate sodium salt (INT-767): Process optimization, synthesis and characterization of metabolites.

Herein we report our synthetic efforts in supporting the development of the bile alcohol sulfate INT-767, a FXR/TGR5 dual agonist with remarkable therapeutic potential for liver disorders. We describe the process development to a final route for large scale preparation and analogues synthesis. Key sequences include Grignard addition, a one-pot two-step shortening-reduction of the carboxylic side chain, and the final sulfation reaction. The necessity for additional steps such as the protection/deprotection of hydroxyl groups at the steroidal body was also evaluated for step-economy and formation of side-products. Critical bottlenecks such as the side chain degradation have been tackled using flow technology before scaling-up individual steps. The final synthetic route may be successfully employed to produce the amount of INT-767 required to support late-stage clinical development of the compound. Furthermore, potential metabolites have been synthesized, characterized and evaluated for their ability to modulate FXR and TGR5 receptors providing key reference standards for future drug investigations, as well as offering further insights into the structure-activity relationships of this class of compounds.

The Stone Guest: How Does pH Affect Binding Properties of PD-1/PD-L1 Inhibitors?

The interaction between programmed cell death-1 (PD-1) and its ligand PD-L1 activates a coinhibitory signal that blocks T-cell activation, promoting the immune escape process in the tumor microenvironment. Development of monoclonal antibodies targeting and inhibiting PD-1/PD-L1 interaction as anticancer immunotherapies has proved successful in multiple clinical settings and for various types of cancer. Notwithstanding, limitations exist with the use of these biologics, including drug resistance and narrow therapeutic response rate in a majority of patients, that demand for the design of more efficacious small molecule-based immunotherapies. Alteration of pH in the tumor microenvironment is a key factor that is involved in promoting drug resistance, tumor survival and progression. In this study, we have investigated the effect of pH shifts on binding properties of distinct classes of PD-L1 inhibitors, including macrocyclic peptide and small molecules. Results expand structure-activity relationships of PD-L1 inhibitors, providing insights into structural features and physicochemical properties that are useful for the design of ligands that may escape a drug resistance mechanism associated to variable pH conditions of tumor microenvironment.

Integrating experimental and computational techniques to study chromatographic enantioresolutions of chiral tetrahydroindazole derivatives.

With the selection of partially saturated 2H-indazoles as model compounds, we demonstrate the possibility to use Whelk-O1 chiral stationary phases (CSPs) to succeed in efficient small-scale preparative enantioseparations. Runs of three consecutive liquid chromatography injections (about 300 µg of racemate repeatedly injected in a 100 µL loop) produced groups of peaks without band contamination (α = 1.2 and RS = 2.57). With this procedure approximately 3.0 mg of each enantiomer, with enantiomeric excess ≥ 97% were obtained. Very profitably, the high volatility of n-hexane used as the sole eluent facilitated the solvent evaporation after the enantiomer recovery. High resolution mass spectrometry analysis confirmed that the chemical identity of the two enantiomers was preserved along the entire process. The ability of Whelk-O1 phases in enantioseparating structurally similar compounds was confirmed with the analysis of other two racemates. Moreover, the relevant chemoselectivity exhibited by the CSP towards the three racemates should allow to simultaneously optimizing the enantioselectivity of different analytes and perform small-scale enantioresolutions of different compounds during the same run. In this study, the integration of experimental off-line electronic circular dichroism analysis with ab initio time-dependent density-functional theory simulations facilitated the assignment of the absolute configuration of the single enantiomers, while a molecular dynamics protocol can be useful to make a priori predictions of the enantioseparation ability of CSP towards selected compounds.

Garcinoic Acid Is a Natural and Selective Agonist of Pregnane X Receptor.

Pregnane X receptor (PXR) is a master xenobiotic-sensing transcription factor and a validated target for immune and inflammatory diseases. The identification of chemical probes to investigate the therapeutic relevance of the receptor is still highly desired. In fact, currently available PXR ligands are not highly selective and can exhibit toxicity and/or potential off-target effects. In this study, we have identified garcinoic acid as a selective and efficient PXR agonist. The properties of this natural molecule as a specific PXR agonist were demonstrated by the screening on a panel of nuclear receptors, the assessment of the physical and thermodynamic binding affinity, and the determination of the PXR-garcinoic acid complex crystal structure. Cytotoxicity, transcriptional, and functional properties were investigated in human liver cells, and compound activity and target engagement were confirmed in vivo in mouse liver and gut tissue. In conclusion, garcinoic acid is a selective natural agonist of PXR and a promising lead compound toward the development of new PXR-regulating modulators.

The Medicinal Chemistry in the Era of Machines and Automation: Recent Advances in Continuous Flow Technology.

Medicinal chemistry plays a fundamental and underlying role in chemical biology, pharmacology, and medicine to discover safe and efficacious drugs. Small molecule medicinal chemistry relies on iterative learning cycles composed of compound design, synthesis, testing, and data analysis to provide new chemical probes and lead compounds for novel and druggable targets. Using traditional approaches, the time from hypothesis to obtaining the results can be protracted, thus limiting the number of compounds that can be advanced into clinical studies. This challenge can be tackled with the recourse of enabling technologies that are showing great potential in improving the drug discovery process. In this Perspective, we highlight recent developments toward innovative medicinal chemistry strategies based on continuous flow systems coupled with automation and bioassays. After a discussion of the aims and concepts, we describe equipment and representative examples of automated flow systems and end-to-end prototypes realized to expedite medicinal chemistry discovery cycles.

Enantioselective HPLC Analysis to Assist the Chemical Exploration of Chiral Imidazolines.

In the present work, we illustrate the ability of high-performance liquid chromatography (HPLC) analysis to assist the synthesis of chiral imidazolines within our medicinal chemistry programs. In particular, a Chiralpak® IB® column containing cellulose tris(3,5-dimethylphenylcarbamate) immobilized onto a 5 µm silica gel was used for the enantioselective HPLC analysis of chiral imidazolines synthesized in the frame of hit-to-lead explorations and designed for exploring the effect of diverse amide substitutions. Very profitably, reversed-phase (RP) conditions succeeded in resolving the enantiomers in nine out of the 10 investigated enantiomeric pairs, with α values always higher than 1.10 and RS values up to 2.31. All compounds were analysed with 50% (v) water while varying the content of the two organic modifiers acetonitrile and methanol. All the employed eluent systems were buffered with 40 mM ammonium acetate while the apparent pH was fixed at 7.5. Based on the experimental results, the prominent role of π-π stacking interactions between the substituted electron-rich phenyl groups outside of the polymeric selector and the complementary aromatic region in defining analyte retention and stereodiscrimination was identified. The importance of compound polarity in explaining the retention behaviour with the employed RP system was readily evident when a quantitative structure-property relationship study was performed on the retention factor values (k) of the 10 compounds, as computed with a 30% (v) methanol containing mobile phase. Indeed, good Pearson correlation coefficients of retention factors (r - log k1st = -0.93; r - log k2nd = -0.94) were obtained with a water solubility descriptor (Ali-logS). Interestingly, a n-hexane/chloroform/ethanol (88:10:2, v/v/v)-based non-standard mobile phase allowed the almost base-line enantioseparation (α = 1.06; RS = 1.26) of the unique compound undiscriminated under RP conditions.

Optimisation by Design of Experiment of Benzimidazol-2-One Synthesis under Flow Conditions.

A novel flow-based approach for the preparation of benzimidazol-2-one (1) scaffold by the 1,1'-carbonyldiimidazole (CDI)-promoted cyclocarbonylation of o-phenylenediamine (2) is reported. Starting from a preliminary batch screening, the model reaction was successfully translated under flow conditions and optimised by means of design of experiment (DoE). The method allowed the efficient preparation of this privileged scaffold and to set up a general protocol for the multigram-scale preparation in high yield, purity, and productivity, and was successfully applied for the multigram flow synthesis of N-(2-chlorobenzyl)-5-cyano-benzimidazol-2-one, which is a key synthon for hit-to-lead explorations in our anti-inflammatory drug discovery program.

Exploiting Chemical Toolboxes for the Expedited Generation of Tetracyclic Quinolines as a Novel Class of PXR Agonists.

The discovery of lead compounds relies on the iterative generation of structure-activity relationship data resulting from the synthesis and biological evaluation of hit analogues. Using traditional approaches, a significant time delay may occur from compound design to results, leading to slow and expensive hit-to-lead explorations. Herein, we have exploited the use of chemical toolboxes to expedite lead discovery and optimization. In particular, the integration of flow synthesizers, automation, process analytical technologies, and computational chemistry has provided a prototype system enabling the multicomponent flow synthesis, in-line analysis, and characterization of chiral tetracyclic quinolines as a novel class of PXR agonists. Within 29 compounds, a novel template 19b (3aS,11R,11aS) was identified with an EC50 of 1.2 µM (efficacy 119%) at the PXR receptor.

Steroids interfere with human carbonic anhydrase activity by using alternative binding mechanisms.

Bile acids have been shown to inhibit human (h) carbonic anhydrases (CA, EC 4.2.1.1) along the gastrointestinal tract, including hCA II. The elucidation of the hormonal inhibition mechanism of the bile acid cholate to hCA II was provided in 2014 by X-ray crystallography. Herein, we extend the inhibition study to a wealth of steroids against four relevant hCA isoforms. Steroids displaying pendants and functional groups of the carboxylate, phenolic or sulfonate types appended at the tetracyclic ring were shown to inhibit the cytosolic CA II and the tumor-associated, transmembrane CA IX in a medium micromolar range (38.9-89.9 µM). Docking studies displayed the different chemotypes CA inhibition mechanisms. Molecular dynamics (MD) gave insights on the stability over time of hyocholic acid binding to CA II.

Binding Mode and Structure-Activity Relationships of ITE as an Aryl Hydrocarbon Receptor (AhR) Agonist.

Discovered as a modulator of the toxic response to environmental pollutants, aryl hydrocarbon receptor (AhR) has recently gained attention for its involvement in various physiological and pathological pathways. AhR is a ligand-dependent transcription factor activated by a large array of chemical compounds, which include metabolites of l-tryptophan (l-Trp) catabolism as endogenous ligands of the receptor. Among these, 2-(1'H-indole-3'-carbonyl)thiazole-4-carboxylic acid methyl ester (ITE) has attracted interest in the scientific community, being endowed with nontoxic, immunomodulatory, and anticancer AhR-mediated functions. So far, no information about the binding mode and interactions of ITE with AhR is available. In this study, we used docking and molecular dynamics to propose a putative binding mode of ITE into the ligand binding pocket of AhR. Mutagenesis studies were then instrumental in validating the proposed binding mode, identifying His 285 and Tyr 316 as important key residues for ligand-dependent receptor activation. Finally, a set of ITE analogues was synthesized and tested to further probe molecular interactions of ITE to AhR and characterize the relevance of specific functional groups in the chemical structure for receptor activity.

Synthesis, physicochemical properties, and biological activity of bile acids 3-glucuronides: Novel insights into bile acid signalling and detoxification.

Glucuronidation is considered an important detoxification pathway of bile acids especially in cholestatic conditions. Glucuronides are less toxic than the parent free forms and are more easily excreted in urine. However, the pathophysiological significance of bile acid glucuronidation is still controversial and debated among the scientific community. Progress in this field has been strongly limited by the lack of appropriate methods for the preparation of pure glucuronides in the amount needed for biological and pharmacological studies. In this work, we have developed a new synthesis of bile acid C3-glucuronides enabling the convenient preparation of gram-scale quantities. The synthesized compounds have been characterized in terms of physicochemical properties and abilities to modulate key nuclear receptors including the farnesoid X receptor (FXR). In particular, we found that C3-glucuronides of chenodeoxycholic acid and lithocholic acid, respectively the most abundant and potentially cytotoxic species formed in patients affected by cholestasis, behave as FXR agonists and positively regulate the gene expression of transporter proteins, the function of which is critical in human conditions related to imbalances of bile acid homeostasis.

Selected cholesterol biosynthesis inhibitors produce accumulation of the intermediate FF-MAS that targets nucleus and activates LXRα in HepG2 cells.

Sterol intermediates of the cholesterol biosynthetic pathway have drawn attention for novel biological activities. Follicular fluid meiosis activating sterol (FF-MAS) is a LXRα ligand and a potential modulator of physiologic processes regulated by nuclear receptors, such as lipid homeostasis and cell proliferation. In this work, we established a model to selectively accumulate FF-MAS in HepG2 cells, by using a combination of the inhibitors AY9944 and 17-hydroxyprogesterone to block C14-sterol reductases and the downstream C4-demethylase complex. We investigated the effects produced by altered levels of cholesterol biosynthesis intermediates, in order to dissect their influence on LXRα signaling. In particular, endogenously accumulated FF-MAS was able to modulate the expression of key genes in cholesterol metabolism, to activate LXRα nuclear signaling resulting in increased lipogenesis, and to inhibit HepG2 cells proliferation. Moreover, a fluorescent ester derivative of FF-MAS localized in nuclear lipid droplets, suggesting a role for these organelles in the storage of signaling lipids interacting with nuclear partners.

Bile acid derivatives as ligands of the farnesoid x receptor: molecular determinants for bile acid binding and receptor modulation.

Bile acids are a peculiar class of steroidal compounds that never cease to amaze. From being simple detergents with a primary role in aiding the absorption of fats and fat-soluble vitamins, bile acids are now widely considered as crucial hormones endowed with genomic and non-genomic functions that are mediated by their interaction with several proteins including the nuclear receptor Farnesoid X Receptor (FXR). Taking advantages of the peculiar properties of bile acids in interacting with the FXR receptor, several biliary derivatives have been synthesized and tested as FXR ligands. The availability of these compounds has contributed to characterize the receptor from a structural, patho-physiological and therapeutic standpoint. Among these, obeticholic acid is a first-in-class FXR agonist that is demonstrating hepatoprotective effects upon FXR activation in patients with liver diseases such as primary biliary cirrhosis and nonalcoholic steatohepatitis. This review provides an historical overview of the rationale behind the discovery of obeticholic acid and chemical tools generated to depict the molecular features and bio-pharmacological relevance of the FXR receptor, as well as to summarize structure-activity relationships of bile acid-based FXR ligands so far reported.

Beyond bile acids: targeting Farnesoid X Receptor (FXR) with natural and synthetic ligands.

The modulation of FXR receptor remains an attractive area in drug discovery to develop novel therapeutic opportunities for liver and metabolic disorders. Despite the large variety of FXR ligands reported so far, only a very restricted number of agonists have entered in clinical settings. In this review article we provide the reader with an overview on the different classes of natural and synthetic ligands that have been developed by academic groups and pharmaceutical companies to target FXR. We discuss their structure-activity relationships, analyzing the binding modes that some of these compounds adopt to interact with the receptor.

Synthesis of atypical bile acids for use as investigative tools for the genetic defect of 3ß-hydroxy-Δ(5)-C27-steroid oxidoreductase deficiency.

Deficiency of 3ß-hydroxy-Δ(5)-C27-steroid oxidoreductase (HSD3B7), an enzyme catalyzing the second step in the pathway for bile acid synthesis, leads to a complete lack of the primary bile acids, cholic and chenodeoxycholic acids, and the accumulation of 3ß,7α-dihydroxy- and 3ß,7α,12α-trihydroxy-Δ(5)-cholenoic acids. Patients affected by this autosomal recessive genetic defect develop cholestatic liver disease that is clinically responsive to primary bile acid therapy. Reference standards of these compounds are needed to facilitate diagnosis and to accurately quantify biochemical responses to therapy. Described are a novel synthesis of atypical bile acids that characterize the HSD3B7 deficiency and their effect on bile acid-activated nuclear receptors, target genes and cytochromes involved in bile acid homeostasis and detoxification. The failure of 3ß-hydroxy-Δ(5)-cholenoic acids to function as FXR, PXR and CAR agonists and to exert hepatoprotective actions explains the mechanism for progressive cholestatic liver disease in patients with HSD3B7 deficiency.