Discovery and Structure-Activity Relationship of Cadazolid: A First-In-Class Quinoxolidinone Antibiotic for the Treatment of Clostridioides difficile Infection.

Clostridioides difficile (C. difficile) is one of the leading causes of healthcare-associated infections worldwide. The increasing incidence of strains resistant to currently available therapies highlights the need for alternative treatment options with a novel mode of action. Oxazolidinones that are connected to a quinolone moiety with a pyrrolidine linker, such as compound 1, are reported to exhibit potent broadspectrum antibacterial activity. In an effort to optimize this class of compounds for the treatment of C. difficile infection (CDI), we have identified cadazolid (9), a first-in-class quinoxolidinone antibiotic, which is a potent inhibitor of C. difficile protein synthesis. In order to achieve narrow-spectrum coverage of clinically most relevant strains without affecting the gut microbiota, an emphasis was placed on abolishing activity against commensals of the intestinal microbiome while retaining good coverage of pathogenic C. difficile, including hypervirulent and epidemic strains.

S1P1 Modulator-Induced G αi Signaling and ß-Arrestin Recruitment Are Both Necessary to Induce Rapid and Efficient Reduction of Blood Lymphocyte Count In Vivo.

S1P1 (sphingosine-1-phosphate receptor 1) agonists prevent lymphocyte egress from secondary lymphoid organs and cause a reduction in the number of circulating blood lymphocytes. We hypothesized that S1P1 receptor modulators with pathway-selective signaling properties could help to further elucidate the molecular mechanisms involved in lymphocyte trapping. A proprietary S1P1 receptor modulator library was screened for compounds with clear potency differences in ß-arrestin recruitment and G protein alpha i subunit (G αi) protein-mediated signaling. We describe here the structure-activity relationships of highly potent S1P1 modulators with apparent pathway selectivity for ß-arrestin recruitment. The most differentiated compound, D3-2, displayed a 180-fold higher potency in the ß-arrestin recruitment assay (EC50 0.9 nM) compared with the G αi-activation assay (167 nM), whereas ponesimod, a S1P1 modulator that is currently in advanced clinical development in multiple sclerosis, was equipotent in both assays (EC50 1.5 and 1.1 nM, respectively). Using these novel compounds as pharmacological tools, we showed that although a high potency in ß-arrestin recruitment is required to fully internalize S1P1 receptors, the potency in inducing G αi signaling determines the rate of receptor internalization in vitro. In contrast to ponesimod, the compound D3-2 did not reduce the number or circulating lymphocytes in rats despite high plasma exposures. Thus, for rapid and maximal S1P1 receptor internalization a high potency in both G αi signaling and ß-arrestin recruitment is mandatory and this translates into efficient reduction of the number of circulating lymphocytes in vivo.

Cenerimod, a novel selective S1P1 receptor modulator with unique signaling properties.

Sphingosine-1-phosphate receptor 1 (S1P1 ) modulators sequester circulating lymphocytes within lymph nodes, thereby preventing potentially pathogenic autoimmune cells from exiting into the blood stream and reaching inflamed tissues. S1P1 receptor modulation may thus offer potential to treat various autoimmune diseases. The first nonselective S1P1-5 receptor modulator FTY720/fingolimod/Gilenya® has successfully demonstrated clinical efficacy in relapsing forms of multiple sclerosis. However, cardiovascular, hepatic, and respiratory side-effects were reported and there is a need for novel S1P1 receptor modulators with better safety profiles. Here, we describe the discovery of cenerimod, a novel, potent and selective S1P1 receptor modulator with unique S1P1 receptor signaling properties and absence of broncho- and vasoconstrictor effects ex vivo and in vivo. Cenerimod dose-dependently lowered circulating lymphocyte counts in rats and mice after oral administration and effectively attenuated disease parameters in a mouse experimental autoimmune encephalitis (EAE) model. Cenerimod has potential as novel therapy with improved safety profile for autoimmune diseases with high unmet medical need.

Synthesis and Characterization of Tetrahydropyran-Based Bacterial Topoisomerase Inhibitors with Antibacterial Activity against Gram-Negative Bacteria.

There is an urgent unmet medical need for novel antibiotics that are effective against a broad range of bacterial species, especially multidrug resistant ones. Tetrahydropyran-based inhibitors of bacterial type II topoisomerases (DNA gyrase and topoisomerase IV) display potent activity against Gram-positive pathogens and no target-mediated cross-resistance with fluoroquinolones. We report our research efforts aimed at expanding the antibacterial spectrum of this class of molecules toward difficult-to-treat Gram-negative pathogens. Physicochemical properties (polarity and basicity) were considered to guide the design process. Dibasic tetrahydropyran-based compounds such as 6 and 21 are potent inhibitors of both DNA gyrase and topoisomerase IV, displaying antibacterial activities against Gram-positive and Gram-negative pathogens (Staphylococcus aureus, Enterobacteriaceae, Pseudomonas aeruginosa, and Acinetobacter baumannii). Compounds 6 and 21 are efficacious in clinically relevant murine infection models.

Novel S1P1 receptor agonists - Part 4: Alkylaminomethyl substituted aryl head groups.

In a previous communication we reported on the discovery of alkylamino pyridine derivatives (e.g. 1) as a new class of potent, selective and efficacious S1P1 receptor (S1PR1) agonists. However, more detailed profiling revealed that this compound class is phototoxic in vitro. Here we describe a new class of potent S1PR1 agonists wherein the exocyclic nitrogen was moved away from the pyridine ring (e.g. 11c). Further structural modifications led to the identification of novel alkylaminomethyl substituted phenyl and thienyl derivatives as potent S1PR1 agonists. These new alkylaminomethyl aryl compounds showed no phototoxic potential. Based on their in vivo efficacy and ability to penetrate the brain, the 5-alkyl-aminomethyl thiophenes appeared to be the most interesting class. Potent and selective S1PR1 agonist 20e, for instance, maximally reduced the blood lymphocyte count (LC) for 24 h after oral administration of 10 mg/kg to rat and its brain concentrations reached >500 ng/g over 24 h.

Novel S1P1 receptor agonists - Part 5: From amino-to alkoxy-pyridines.

In a previous communication we reported on the discovery of aminopyridine 1 as a potent, selective and orally active S1P1 receptor agonist. More detailed studies revealed that this compound is phototoxic in vitro. As a result of efforts aiming at eliminating this undesired property, a series of alkoxy substituted pyridine derivatives was discovered. The photo irritancy factor (PIF) of these alkoxy pyridines was significantly lower than the one of aminopyridine 1 and most compounds were not phototoxic. Focused SAR studies showed, that 2-, 3-, and 4-pyridine derivatives delivered highly potent S1P1 receptor agonists. While the 2-pyridines were clearly more selective against S1PR3, the corresponding 3- or 4-pyridine analogues showed significantly longer oral half-lives and as a consequence longer pharmacological duration of action after oral administration. One of the best compounds, cyclopentoxy-pyridine 45b lacked phototoxicity, showed EC50 values of 0.7 and 140 nM on S1PR1 and S1PR3, respectively, and maximally reduced the blood lymphocyte count for at least 24 h after oral administration of 10 mg/kg to Wistar rats.

Physiologically-Based Pharmacokinetic Modeling of Macitentan: Prediction of Drug-Drug Interactions.

INTRODUCTION: Macitentan is a novel dual endothelin receptor antagonist for the treatment of pulmonary arterial hypertension (PAH). It is metabolized by cytochrome P450 (CYP) enzymes, mainly CYP3A4, to its active metabolite ACT-132577. METHODS: A physiological-based pharmacokinetic (PBPK) model was developed by combining observations from clinical studies and physicochemical parameters as well as absorption, distribution, metabolism and excretion parameters determined in vitro. RESULTS: The model predicted the observed pharmacokinetics of macitentan and its active metabolite ACT-132577 after single and multiple dosing. It performed well in recovering the observed effect of the CYP3A4 inhibitors ketoconazole and cyclosporine, and the CYP3A4 inducer rifampicin, as well as in predicting interactions with S-warfarin and sildenafil. The model was robust enough to allow prospective predictions of macitentan-drug combinations not studied, including an alternative dosing regimen of ketoconazole and nine other CYP3A4-interacting drugs. Among these were the HIV drugs ritonavir and saquinavir, which were included because HIV infection is a known risk factor for the development of PAH. CONCLUSION: This example of the application of PBPK modeling to predict drug-drug interactions was used to support the labeling of macitentan (Opsumit).

Novel tetrahydropyran-based bacterial topoisomerase inhibitors with potent anti-gram positive activity and improved safety profile.

Novel antibacterial drugs that are effective against infections caused by multidrug resistant pathogens are urgently needed. In a previous report, we have shown that tetrahydropyran-based inhibitors of bacterial type II topoisomerases (DNA gyrase and topoisomerase IV) display potent antibacterial activity and exhibit no target-mediated cross-resistance with fluoroquinolones. During the course of our optimization program, lead compound 5 was deprioritized due to adverse findings in cardiovascular safety studies. In the effort of mitigating these findings and optimizing further the pharmacological profile of this class of compounds, we have identified a subseries of tetrahydropyran-based molecules that are potent DNA gyrase and topoisomerase IV inhibitors and display excellent antibacterial activity against Gram positive pathogens, including clinically relevant resistant isolates. One representative of this class, compound 32d, elicited only weak inhibition of hERG K(+) channels and hNaV1.5 Na(+) channels, and no effects were observed on cardiovascular parameters in anesthetized guinea pigs. In vivo efficacy in animal infection models has been demonstrated against Staphylococcus aureus and Streptococcus pneumoniae strains.

Novel S1P(1) receptor agonists--part 3: from thiophenes to pyridines.

In preceding communications we summarized our medicinal chemistry efforts leading to the identification of potent, selective, and orally active S1P1 agonists such as the thiophene derivative 1. As a continuation of these efforts, we replaced the thiophene in 1 by a 2-, 3-, or 4-pyridine and obtained less lipophilic, potent, and selective S1P1 agonists (e.g., 2) efficiently reducing blood lymphocyte count in the rat. Structural features influencing the compounds' receptor affinity profile and pharmacokinetics are discussed. In addition, the ability to penetrate brain tissue has been studied for several compounds. As a typical example for these pyridine based S1P1 agonists, compound 53 showed EC50 values of 0.6 and 352 nM for the S1P1 and S1P3 receptor, respectively, displayed favorable PK properties, and penetrated well into brain tissue. In the rat, compound 53 maximally reduced the blood lymphocyte count for at least 24 h after oral dosing of 3 mg/kg.

Novel S1P(1) receptor agonists--part 2: from bicyclo[3.1.0]hexane-fused thiophenes to isobutyl substituted thiophenes.

Previously, we reported on the discovery of a novel series of bicyclo[3.1.0]hexane fused thiophene derivatives that serve as potent and selective S1P1 receptor agonists. Here, we discuss our efforts to simplify the bicyclohexane fused thiophene head. In a first step the bicyclohexane moiety could be replaced by a simpler, less rigid cyclohexane ring without compromising the S1P receptor affinity profile of these novel compounds. In a second step, the thiophene head was simplified even further by replacing the cyclohexane ring with an isobutyl group attached either to position 4 or position 5 of the thiophene. These structurally much simpler headgroups again furnished potent and selective S1P1 agonists (e.g., 87), which efficiently and dose dependently reduced the number of circulating lymphocytes upon oral administration to male Wistar rats. For several compounds discussed in this report lymphatic transport is an important route of absorption that may offer opportunities for a tissue targeted approach with minimal plasma exposure.

Novel S1P1 receptor agonists--part 1: From pyrazoles to thiophenes.

From a high-throughput screening campaign aiming at the identification of novel S1P1 receptor agonists, the pyrazole derivative 2 emerged as a hit structure. Medicinal chemistry efforts focused not only on improving the potency of the compound but in particular also on resolving its inherent instability issue. This led to the discovery of novel bicyclo[3.1.0]hexane fused thiophene derivatives. Compounds with high affinity and selectivity for S1P1 efficiently reducing the blood lymphocyte count in the rat were identified. For instance, compound 85 showed EC50 values of 7 and 2880 nM on S1P1 and S1P3, respectively, had favorable pharmacokinetic properties in rat and dog, distributed well into brain tissue, and efficiently and dose dependently reduced the blood lymphocyte count in the rat. After oral administration to spontaneously hypertensive rats, the S1P1 selective compound 85 showed no effect on mean arterial blood pressure and affected the heart rate during the wake phase of the animals only.

Design, synthesis, and characterization of novel tetrahydropyran-based bacterial topoisomerase inhibitors with potent anti-gram-positive activity.

There is an urgent need for new antibacterial drugs that are effective against infections caused by multidrug-resistant pathogens. Novel nonfluoroquinolone inhibitors of bacterial type II topoisomerases (DNA gyrase and topoisomerase IV) have the potential to become such drugs because they display potent antibacterial activity and exhibit no target-mediated cross-resistance with fluoroquinolones. Bacterial topoisomerase inhibitors that are built on a tetrahydropyran ring linked to a bicyclic aromatic moiety through a syn-diol linker show potent anti-Gram-positive activity, covering isolates with clinically relevant resistance phenotypes. For instance, analog 49c was found to be a dual DNA gyrase-topoisomerase IV inhibitor, with broad antibacterial activity and low propensity for spontaneous resistance development, but suffered from high hERG K(+) channel block. On the other hand, analog 49e displayed lower hERG K(+) channel block while retaining potent in vitro antibacterial activity and acceptable frequency for resistance development. Furthermore, analog 49e showed moderate clearance in rat and promising in vivo efficacy against Staphylococcus aureus in a murine infection model.

Structure-guided design, synthesis and biological evaluation of novel DNA ligase inhibitors with in vitro and in vivo anti-staphylococcal activity.

A series of 2-amino-[1,8]-naphthyridine-3-carboxamides (ANCs) with potent inhibition of bacterial NAD(+)-dependent DNA ligases (LigAs) evolved from a 2,4-diaminopteridine derivative discovered by HTS. The design was guided by several highly resolved X-ray structures of our inhibitors in complex with either Streptococcus pneumoniae or Escherichia coli LigA. The structure-activity-relationship based on the ANC scaffold is discussed. The in-depth characterization of 2-amino-6-bromo-7-(trifluoromethyl)-[1,8]-naphthyridine-3-carboxamide, which displayed promising in vitro (MIC Staphylococcus aureus 1 mg/L) and in vivo anti-staphylococcal activity, is presented.

The selective sphingosine 1-phosphate receptor 1 agonist ponesimod protects against lymphocyte-mediated tissue inflammation.

Lymphocyte exit from lymph nodes and their recirculation into blood is controlled by the sphingolipid sphingosine 1-phosphate (S1P). The cellular receptor mediating lymphocyte exit is S1P(1), one of five S1P receptors. Nonselective agonists for S1P receptors lead to blood lymphocyte count reduction. The effects of selective S1P(1) agonists on blood lymphocyte count and their impact in models of lymphocyte-mediated tissue inflammation have been less investigated. We describe here the general pharmacology of ponesimod, (Z,Z)-5-[3-chloro-4-((2R)-2,3-dihydroxy-propoxy)-benzylidene]-2-propylimino-3-o-tolyl-thiazolidin-4-one, a new, potent, and orally active selective S1P(1) agonist. Ponesimod activated S1P(1)-mediated signal transduction with high potency (EC(50) of 5.7 nM) and selectivity. Oral administration of ponesimod to rats led to a dose-dependent decrease of blood lymphocyte count. After discontinuation of dosing, blood lymphocyte count returned to baseline within 48 h. Ponesimod prevented edema formation, inflammatory cell accumulation, and cytokine release in the skin of mice with delayed-type hypersensitivity. Ponesimod also prevented the increase in paw volume and joint inflammation in rats with adjuvant-induced arthritis. These data show that selective activation of S1P(1) using ponesimod leads to blood lymphocyte count reduction and efficacy in models of lymphocyte-mediated tissue inflammation. Immunomodulation with a rapidly reversible S1P(1)-selective agonist may represent a new therapeutic approach in lymphocyte-mediated autoimmune diseases.

2-imino-thiazolidin-4-one derivatives as potent, orally active S1P1 receptor agonists.

Sphingosine-1-phosphate (S1P) is a widespread lysophospholipid which displays a wealth of biological effects. Extracellular S1P conveys its activity through five specific G-protein coupled receptors numbered S1P(1) through S1P(5). Agonists of the S1P(1) receptor block the egress of T-lymphocytes from thymus and lymphoid organs and hold promise for the oral treatment of autoimmune disorders. Here, we report on the discovery and detailed structure-activity relationships of a novel class of S1P(1) receptor agonists based on the 2-imino-thiazolidin-4-one scaffold. Compound 8bo (ACT-128800) emerged from this series and is a potent, selective, and orally active S1P(1) receptor agonist selected for clinical development. In the rat, maximal reduction of circulating lymphocytes was reached at a dose of 3 mg/kg. The duration of lymphocyte sequestration was dose dependent. At a dose of 100 mg/kg, the effect on lymphocyte counts was fully reversible within less than 36 h. Pharmacokinetic investigation of 8bo in beagle dogs suggests that the compound is suitable for once daily dosing in humans.

Transporters--the view from industry.

The involvement of transport proteins in the disposition of drugs is receiving much attention of the scientific community. Recently, researchers from academia have surmised that drug transport rather than passive diffusion is the regular mechanism for molecules to cross cell membranes. On bare face value, however, sound evidence of the impact of transport proteins on clinical pharmacokinetics has been a trickle rather than a stream of convincing studies during the last decade, in stark contrast to the number of in vitro studies published. Progress in this area may have been impeded by a number of factors. Only a limited number of small-molecule drugs fall within the physicochemical property space (i.e., high hydrophilicity and low passive permeability) that makes them predestined as transport protein substrates without other pharmacokinetic processes (e.g., passive diffusion, metabolism, nonspecific binding to tissue proteins) blurring the picture. The vast majority of drug molecules are lipophilic enough to be amenable to passive diffusion across cell membranes and to undergo metabolism to some extent. In these cases, clinical evidence relies heavily on the observation of pharmacokinetic drug-drug interactions not readily explained by the interference with drug metabolizing enzymes. Given the circumstances outlined above, it is not surprising that, based upon clinical observations, the final assessment as to the overall relevance of drug transport for clinical pharmacokinetics is still pending.

Cyclization of the acyl glucuronide metabolite of a neutral endopeptidase inhibitor to an electrophilic glutarimide: synthesis, reactivity, and mechanistic analysis.

The neutral endopeptidase inhibitor (2R)-2-[(1-{[(5-ethyl-1,3,4-thiadiazol-2-yl)amino]carbonyl}cyclopentyl)methyl]pentanoic acid 2 is metabolized to acyl glucuronide 3. Unprecedentedly, at pH 7.4, 3 does not undergo the O-acyl migration characteristic of acyl glucuronides but rapid, eliminative cyclization (t1/2 at 37 degrees C, 10.2 min) to glutarimide 4. Glucuronide 3 was synthesized efficiently via acylation of benzylglucuronate with N-benzyloxymethyl-protected 2. Glucuronide and imide reacted rapidly in aqueous solution, pH 7.4, with amino acids and glutathione to form stable amides and unstable thioesters. Imide 4 acylated eight lysine Nepsilon-amino groups of human serum albumin. Rapid cyclization of 3 was attributed to attack on the ester linkage by an unusually nucleophilic glutaramide NH (pKa in 2 = 9.76). N-propyl 3 was refractory to acyl migration and cyclization. This suggested a synthetic strategy for preparing analogues of 2 that form chemically stable acyl glucuronides.

Determination of degradation pathways and kinetics of acyl glucuronides by NMR spectroscopy.

Acyl glucuronides have been implicated in the toxicity of many xenobiotics and marketed drugs. These toxicities are hypothesized to be a consequence of covalent binding of the reactive forms of the acyl glucuronide to proteins. Reactive intermediates of the acyl glucuronide arise from the migration of the aglycone leading to other positional and stereoisomers under physiological conditions. In order to screen for the potential liabilities of these metabolites during the early phase of pharmaceutical development, an NMR method based on the disappearance of the anomeric resonance of the O-1-acyl glucuronide was used to monitor the degradation kinetics of 11 structurally diverse acyl glucuronides, including those produced from the known nonsteroidal anti-inflammatory drugs (NSAIDs). The acyl glucuronides were either chemically synthesized or were isolated from biological matrices (bile, urine, and liver microsomal extracts). The half-lives attained utilizing this method were found to be comparable to those reported in the literature. NMR analysis also enabled the delineation of the two possible pathways of degradation: acyl migration and hydrolytic cleavage. The previously characterized 1H resonances of acyl migrated products are quite distinguishable from those that arise from hydrolysis. The NMR method described here could be used to rank order acyl glucuronide forming discovery compounds based on the potential reactivity of the conjugates and their routes of decomposition under physiological conditions. Furthermore, we have shown that in vitro systems such as liver microsomal preparations can be used to generate sufficient quantities of acyl glucuronides from early discovery compounds for NMR characterization. This is particularly important, as we often have limited supply of early discovery compounds to conduct in vivo studies to generate sufficient quantities of acyl glucuronides for further characterization.

Novel selective inhibitors of neutral endopeptidase for the treatment of female sexual arousal disorder.

A series of substituted glutaramides were synthesised using Candoxatrilat 1 as a lead and evaluated for potency against neutral endopeptidase (NEP) as a potential treatment for female sexual arousal disorder (FSAD). In this paper, we describe studies in which we were able to increase NEP activity substantially over the levels reported for previous compounds from this programme by appropriate substitution in both the P(1)(') and P(2)(') regions. Optimisation led to the 4-chlorophenpropylamide S-30 which was selected as a candidate for further study.