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Saliva is an attractive sampling matrix for measuring various endogenous and exogeneous substances but requires sample treatment prior to chromatographic analysis. Exploiting supercritical CO2 for both extraction and chromatography simplifies sample preparation, reduces organic solvent consumption, and minimizes exposure to potentially infectious samples, but has not yet been applied to oral fluid. Here, we demonstrate the feasibility and benefits of online supercritical fluid extraction coupled to supercritical fluid chromatography and single-quadrupole mass spectrometry for monitoring the model salivary tracer caffeine. A comparison of 13 C- and 32 S-labeled internal standards with external standard calibration confirmed the superiority of stable isotope-labeled caffeine over nonanalogous internal standards. As proof of concept, the validated method was applied to saliva from a magnetic resonance imaging study of gastric emptying. After administration of 35 mg caffeine via ice capsule, salivary levels correlated with magnetic resonance imaging data, corroborating caffeine's usefulness as tracer of gastric emptying (R2 = 0.945). In contrast to off-line methods, online quantification required only minute amounts of organic solvents and a single manual operation prior to online bioanalysis of saliva, thus demonstrating the usefulness of CO2 -based extraction and separation techniques for potentially infective biomatrices.

The pathogenesis of autoimmune complications triggered by SARS‐CoV2 has not been completely elucidated. Here, we performed an analysis of the cellular immune status, cell ratios, and monocyte populations of patients with COVID‐19 treated in the intensive care unit (ICU) (cohort 1, N = 23) and normal care unit (NCU) (cohort 2, n = 10) compared with control groups: patients treated in ICU for noninfectious reasons (cohort 3, n = 30) and patients treated in NCU for infections other than COVID‐19 (cohort 4, n = 21). Patients in cohort 1 presented significant differences in comparison with the other cohorts, including reduced frequencies of lymphocytes, reduced CD8+T‐cell count, reduced percentage of activated and intermediate monocytes and an increased B/T8 cell ratio. Over time, patients in cohort 1 who died presented with lower counts of B, T, CD4+T, CD8+T‐lymphocytes, NK cells, and activated monocytes. The B/T8 ratio was significantly lower in the group of survivors. In cohort 1, significantly higher levels of IgG1 and IgG3 were found, whereas cohort 3 presented higher levels of IgG3 compared to controls. Among many immune changes, an elevated B/T8‐cell ratio and a reduced rate of activated monocytes were mainly observed in patients with severe COVID‐19. Both parameters were associated with death in cohort 1., Patients with severe COVID‐19 present a particular set of immune changes in comparison to patients with mild disease and controls. These include consumption of certain monocyte and lymphocyte populations and an elevated B/T8 Ratio. COVID‐19 patients in general share a proinflammatory immunoglobulin profile with elevated proportions of IgG1 and/or IgG3.

We engineered the cytochrome P450 monooxygenase CYP107D1 (OleP) from Streptomyces antibioticus for the stereo‐ and regioselective 7β‐hydroxylation of lithocholic acid (LCA) to yield ursodeoxycholic acid (UDCA). OleP was previously shown to hydroxylate testosterone at the 7β‐position but LCA is exclusively hydroxylated at the 6β‐position, forming murideoxycholic acid (MDCA). Structural and 3DM analysis, and molecular docking were used to identify amino acid residues F84, S240, and V291 as specificity‐determining residues. Alanine scanning identified S240A as a UDCA‐producing variant. A synthetic “small but smart” library based on these positions was screened using a colorimetric assay for UDCA. We identified a nearly perfectly regio‐ and stereoselective triple mutant (F84Q/S240A/V291G) that produces 10‐fold higher levels of UDCA than the S240A variant. This biocatalyst opens up new possibilities for the environmentally friendly synthesis of UDCA from the biological waste product LCA., We report engineering of a P450 monooxygenase for the stereo‐ and regioselective 7β‐hydroxylation of lithocholic acid to produce ursodeoxycholic acid (UDCA). Structural and 3DM analysis, and molecular docking, identified selectivity‐influencing residues. A “small but smart” mutant library was then screened with a selective colorimetric assay. The best mutant has nearly perfect regio‐ and stereoselectivity, enabling a new route for UDCA synthesis.

Methylation of free hydroxyl groups is an important modification for flavonoids. It not only greatly increases absorption and oral bioavailability of flavonoids, but also brings new biological activities. Flavonoid methylation is usually achieved by a specific group of plant O‐methyltransferases (OMTs) which typically exhibit high substrate specificity. Here we investigated the effect of several residues in the binding pocket of the Clarkia breweri isoeugenol OMT on the substrate scope and regioselectivity against flavonoids. The mutation T133M, identified as reported in our previous publication, increased the activity of the enzyme against several flavonoids, namely eriodictyol, naringenin, luteolin, quercetin and even the isoflavonoid genistein, while a reduced set of amino acids at positions 322 and 326 affected both, the activity and the regioselectivity of the methyltranferase. On the basis of this work, methylated flavonoids that are rare in nature were produced in high purity.

Human histone demethylases are known to play an important role in the development of several tumor types. Consequently, they have emerged as important medical targets for the treatment of human cancer. Herein, structural studies on tetrazolylhydrazide inhibitors as a new scaffold for a certain class of histone demethylases, the JmjC proteins, are reported. A series of compounds are structurally described and their respective binding modes to the KDM4D protein, which serves as a high‐resolution model to represent the KDM4 subfamily in crystallographic studies, are examined. Similar to previously reported inhibitors, the compounds described herein are competitors for the natural KDM4 cofactor, 2‐oxoglutarate. The tetrazolylhydrazide scaffold fills an important gap in KDM4 inhibition and newly described, detailed interactions of inhibitor moieties pave the way to the development of compounds with high target‐binding affinity and increased membrane permeability, at the same time., Bring on the subs! Tetrazolylhydrazide compounds are isosteres of 2‐oxoglutarate, which is the natural cofactor of KDM4 proteins. It is believed that substitution of the carboxylic groups with tetrazole and hydrazide moieties will lead to KDM4 inhibitors with improved physicochemical properties. The binding mode of various such ligands in the active site of KDM4D is elucidated and movements of the interacting amino acid residues upon ligand binding are described.

The potassium channel openers flupirtine and retigabine have proven to be valuable analgesics or antiepileptics. Their recent withdrawal due to occasional hepatotoxicity and tissue discoloration, respectively, leaves a therapeutic niche unfilled. Metabolic oxidation of both drugs gives rise to the formation of electrophilic quinones. These elusive, highly reactive metabolites may induce liver injury in the case of flupirtine and blue tissue discoloration after prolonged intake of retigabine. We examined which structural features can be altered to avoid the detrimental oxidation of the aromatic ring and shift oxidation toward the formation of more benign metabolites. Structure-activity relationship studies were performed to evaluate the KV 7.2/3 channel opening activity of 45 derivatives. Sulfide analogues were identified that are devoid of the risk of quinone formation, but possess potent KV 7.2/3 opening activity. For example, flupirtine analogue 3-(3,5-difluorophenyl)-N-(6-(isobutylthio)-2-(pyrrolidin-1-yl)pyridin-3-yl)propanamide (48) has 100-fold enhanced activity (EC50 =1.4 nm), a vastly improved toxicity/activity ratio, and the same efficacy as retigabine in vitro.

Reactive oxygen species (ROS) are known to trigger drug release from arylboronate-containing ROS-responsive prodrugs. In cancer cells, elevated levels of ROS can be exploited for the selective activation of prodrugs via Baeyer-Villiger type oxidation rearrangement sequences. Here, we report a proof of concept to demonstrate that these cascades can as well be initiated by cold physical plasma (CPP). An analog of a recently reported fluorouracil prodrug based on the less toxic drug 5-fluorocytosine (5-FC) was synthesized with a view to laboratory safety reasons and used as a model compound to prove our hypothesis that CPP is suitable as a trigger for the prodrug activation. Although the envisioned oxidation and rearrangement with successive loss of boronic acid species could be achieved by plasma treatment, the anticipated spontaneous liberation of 5-FC was inefficient in the model case. However, the obtained results suggest that custom-tailored CPP-responsive prodrugs might become an evolving research field.

The JumonjiC-domain-containing histone demethylase 2A (JMJD2A, KDM4A) is a key player in the epigenetic regulation of gene expression. Previous publications have shown that both elevated and lowered enzyme levels are associated with certain types of cancer, and therefore the definite role of KDM4A in oncogenesis remains elusive. To identify a novel molecular starting point with favorable physicochemical properties for the investigation of the physiological role of KDM4A, we screened a number of molecules bearing an iron-chelating moiety by using two independent assays. In this way, we were able to identify 2-(1H-tetrazol-5-yl)acetohydrazide as a novel fragment-like lead structure with low relative molecular mass (Mr =142 Da), low complexity, and an IC50 value of 46.6 μm in a formaldehyde dehydrogenase (FDH)-coupled assay and 2.4 μm in an antibody-based assay. Despite its small size, relative selectivity against two other demethylases could be demonstrated for this compound. This is the first example of a tetrazole group as a warhead in JMJD demethylases.

Aims Angiopoietin-2 (Ang-2) is a mediator of capillary leakage, and increased Ang-2 levels were associated with poor in-hospital outcome in a pilot study in cardiogenic shock (CS). In this larger study, we followed this hypothesis and aimed at assessing the predictive role of Ang-2 on short- and long-term mortality, investigating the effect of intra-aortic balloon pump (IABP) treatment on Ang-2 levels, and identifying clinical and procedural predictors of increased Ang-2. Methods and results In the IABP-SHOCK II-trial, 600 patients with CS complicating acute myocardial infarction were assigned to therapy with or without IABP. This substudy included 189 randomized patients with serial blood sampling performed at days 1, 2, and 3. No significant differences in Ang-2 levels were found between patients with or without IABP. The Ang-2 levels above the median at day 1 were associated with 30-day [hazard ratio (HR) 1.98, 95% confidence interval (CI) 1.26–3.10, P = 0.002) and 1-year mortality (HR 2.21, 95% CI 1.49–3.27, P < 0.001). Stratification of patients according to Ang-2 levels at day 3 increased these associations (30 days HR 5.15, 95% CI 2.80–9.45, P < 0.001; 1 year HR 5.24, 95% CI 3.19–8.58, P < 0.001). The Ang-2 concentrations were independent predictors for mortality in multivariate analysis (30 days HR 4.82, 95% CI 1.52–15.23, P = 0.007; 1 year HR 2.01, 95%CI 1.24–3.24, P = 0.005). Predictors of increased Ang-2 levels at day 3 were baseline Ang-2, development of acute kidney injury, bleeding events or transfusion, and impaired reperfusion. Conclusion In CS, high levels of Ang-2 are independently associated with poor short- and long-term outcome and associated with the reperfusion success as well as complications. Clinical Trial Registration URL: www.clinicaltrials.gov; unique identifier: NCT00491036

Patients with cardiogenic shock have a very high mortality. Here we report the first use of a percutaneous pulsatile cardiac assist device, based on a diagonal pump synchronized with the heart cycle by means of an electrocardiographic signal in adult pigs. Eight domestic pigs underwent mandatory ventilation. During sinus rhythm, there were no differences between pulsatile and nonpulsatile perfusion with regard to pulmonary artery pressure, pulmonary wedge pressure, central venous pressure, mean arterial pressure (MAP), mean pulse pressure, and mean coronary artery flow (CAF). After 2 min of complete cardiac arrest (ventricular fibrillation), circulatory support with the i-cor in venoarterial nonpulsatile extracorporeal membrane oxygenation (ECMO) mode (3 L/min) restored systemic circulation, with an increase of MAP to 78.3 mm Hg and CAF to 5.27 mL/min. After changing from ECMO settings to pulsatile mode (3 L/min, 75 bpm, pulse amplitude range 3500 rpm), MAP did not change significantly (75.6 mm Hg); however, CAF increased to 8.45 mL/min. After changing back to nonpulsatile mode, MAP remained stable (83.6 mm Hg), but CAF decreased to 4.85 mL/min. Thereafter, pulsatile cardiac assist was established with a reduced blood flow of 2.5 L/min, and the pulse amplitude range was extended to 4500 rpm. Under these conditions, MAP remained stable (71.0 mm Hg), but CAF significantly increased to 15.2 mL/min (P

Openers of neuronal voltage-gated potassium channels (KV ) are of interest as therapeutic agents for treating pain (flupirtine) and epilepsy (retigabine). In an effort to better understand the mechanisms of action and toxicity of flupirtine, we synthesized nine novel analogues with varying redox behavior. Flupirtine can be oxidatively metabolized into azaquinone diimines; thus, the oxidation potentials of flupirtine and its analogues were measured by cyclic voltammetry. KV 7.2/3 (KCNQ2/3) opening activity was determined by an established assay with HEK293 cells overexpressing these channels. A link was found between the oxidation potentials of the compounds and their EC50 values for potassium channel opening activity. On the other hand, no correlation was observed between oxidation potentials and cytotoxicity in cultures of transgenic mouse hepatocytes (TAMH). These results support the idea that oxidative metabolites of flupirtine contribute to the mechanism of action, similar to what was recently proposed for acetaminophen (paracetamol), but not to hepatotoxicity.

Racemic misonidazole, a radiosensitizer formally used in radiation therapy of cancer and to date still applied, was once reported to exhibit strong inhibitory effects on mouse glutathione peroxidases (GPX). This appeared to qualify misonidazole as a lead structure for the development of novel GPX inhibitors to cause oxidative stress in chemotherapy-resistant tumors. A unique feature of misonidazole as an inhibitor of GPX is the absence of a thiol functionality. Therefore, it was expected to selectively target inhibition devoid of promiscuous interactions with cations and sulfhydryl groups. We synthesized the isomers of misonidazole and analyzed the ability of chiroptical high-performance liquid chromatography (HPLC) to identify the particular enantiomers. Due to the chiral pool synthesis, the assignment of the correct configuration could be verified. Finally, we evaluated both isomers for their inhibitory activities on bovine erythrocyte GPx-1, which is 87% homologous to the human enzyme. Despite the previously reported inhibition of racemic misonidazole on the less homologous mouse GPx-1, we did not find any significant inhibitory activity on the bovine enzyme for either isomer. Though misonidazole appears unlikely to be an inhibitor of human GPx-1 activity, we still spotlight misonidazole as a promising fragment-like lead structure in general.

Aromatic heterocycles with basic nitrogen atoms as well as carboxylic acid derivatives are the dominating chemical space in the universe of drug-like molecules. These established and exceedingly evaluated structural motifs have to be combined with elements of diversity in order to chart less well-explored galaxies of chemical space and to be able to tackle seemingly undruggable targets. Flat scaffolds should be replaced by shapely molecular cores. In this context, it has been unheeded that phenyl rings in diaryl sulfides are less co-planar than in ethers and that the metabolic interconnection of sulfides and sulfoxides offers advantages that are unalike from the chemistry of amines and N-oxides in the CHN-O world. Moreover, σ-hole potentials increase with the polarizability of the atom N < P < O < S and do not only play a role in long-time overlooked halogen bonds. Examples for λ2 , λ4 , and λ6 S-based functionalities related to improved solubility, reduced drug resistance, linkers in drug conjugates, drug-targeting to parasites, and as basis for drug monitoring in sports are given and discussed.

Despite their simplicity, relatively few examples of 1,2,4 (1,3,4)-amino-, azido-, and hydroxy-substituted cyclopentanes are reported in the literature. We found that cyclopent-3-en-1-ol can be transformed into a significant variety of compounds of this class by relatively common and efficient synthetic procedures. Stereochemical control of epoxidation of the cyclopentene double bond can be achieved by varying the substitutents at C4. The C4 substituent and epoxide functional group can be converted into a variety of intermediates with differential protection designed for use in applications requiring regiospecific control for further elaboration of the cyclopentane scaffold.

NO-Donatoren haben aufgrund ihrer hamodynamischen Wirkungen positive Effekte bei Patienten mit Herzinsuffizienz. Zur Therapie der akuten Herzinsuffizienz sind Glyceroltrinitrat, Isosorbiddinitrat und Natrium-Nitroprussid etabliert. Sie fuhren durch eine Reduktion der Vor- und Nachlast zu einer Stabilisierung des Patienten. Aufgrund der Toleranzentwicklung eignen sich NO-Donatoren nur eingeschrankt zur Langzeittherapie. Bei der Behandlung der chronischen Herzinsuffizienz wird aktuell nur eine Kombinationstherapie aus Isosorbiddinitrat und Hydralazin in besonderen Situationen empfohlen. Das Nitrat Pentaerythrityltetranitrat nimmt aufgrund einer verminderten Toleranzentwicklung eine besondere Stellung ein. Die Wirkung dieses Medikaments bei Patienten mit chronischer Herzinsuffizienz und sekundarer pulmonaler Hypertonie wird aktuell in klinischen Studien untersucht.

The conversion of 1-deoxy-D-xylulose-5-phosphate (DOXP) to 2-C-methyl-D-erythritol-4-phosphate (MEP) is effectively blocked by 1-deoxy-D-xylulose-5-phosphate reductoisomerase (Dxr) inhibitors such as the natural antibiotic fosmidomycin. Prediction of binding affinities for closely related Dxr ligands as well as estimation of the affinities of structurally more distinct inhibitors within this class of non-hydrolyzable phosphate mimics relies on the synthesis of fosmidomycin derivatives with a broad range of target affinity. Maintaining the phosphonic acid moiety, linear modifications of the lead structure were carried out in an effort to expand the SAR of this physicochemically challenging class of compounds. Synthetic access to a set of phosphonic acids with inhibitory activity (IC(50)) in the range from 1 to >30 microM vs. E. coli Dxr and 0.4 to 20 microM against P. falciparum Dxr is reported.

In order to commemorate Carl Mannich's achievement with many implications in the fields of drug design and synthesis, stability of pharmaceutical preparations, and natural product chemistry, we invited researchers to submit their work related to Mannich's pioneering article at the occasion of its centennial anniversary to the Archiv der Pharmazie - where else?

Nonpolar derivatives of heterocyclic aromatic screening hits like the non-selective sirtuin inhibitor splitomicin tend to be poorly soluble in biological fluids. Unlike sp3-rich natural products, flat aromatic compounds are prone to stacking and often difficult to optimize into leads with activity in cellular systems. The aim of this work was to identify anchor points for the introduction of sp3-rich fragments with polar functional groups into the newly discovered active (IC50 = 5 μM) but nonpolar scaffold 1,2-dihydro-3H-naphth[1,2-e][1,3]oxazine-3-thione by a molecular modeling approach. Docking studies were conducted with structural data from crystallized human SIRT2 enzyme. Subsequent evaluation of the in silico hypotheses through synthesis and biological evaluation of the designed structures was accomplished with the aim to discover new SIRT2 inhibitors with improved aqueous solubility. Derivatives of 8-bromo-1,2-dihydro-3H-naphth[1,2-e][1,3]oxazine-3-thione N-alkylated with a hydrophilic morpholino-alkyl chain at the thiocarbamate group intended for binding in the acetyl-lysine pocket of the enzyme appeared to be promising. Both the sulfur of the thiocarbamate and the bromo substituent were assumed to result in favorable hydrophobic interactions and the basic morpholino-nitrogen was predicted to build a hydrogen bond with the backbone Ile196. While the brominated scaffold showed moderately improved activity (IC50 = 1.8 μM), none of the new compounds displayed submicromolar activity. Synthesis and characterization of the new compounds are reported and the possible reasons for the outcome are discussed.

In order to investigate structure-activity relationships in the antiproliferative darpone series, the derivatives 8a and 8b lacking the aryl substituents in either 2- or 4-position were synthesized and evaluated for cancer cell growth inhibition. Since both 8a and 8b were devoid of noteworthy antiproliferative activity, both aryl substituents can be considered as part of the darpone pharmacophore.

Paullones constitute a new family of benzazepinones with promising antitumoral properties. They were recently described as potent, ATP-competitive, inhibitors of the cell cycle regulating cyclin-dependent kinases (CDKs). We here report that paullones also act as very potent inhibitors of glycogen synthase kinase-3b (GSK-3b) (IC50: 4‐80 nm) and the neuronal CDK5/p25 (IC50: 20‐200 nm). These two enzymes are responsible for most of the hyperphosphorylation of the microtubule-binding protein tau, a feature observed in the brains of patients with Alzheimer’s disease and other neurodegenerative ‘taupathies’. Alsterpaullone, the most active paullone, was demonstrated to act by competing with ATP for binding to GSK-3b. Alsterpaullone inhibits the phosphorylation of tau in vivo at sites which are typically phosphorylated by GSK-3b in Alzheimer’s disease. Alsterpaullone also inhibits the CDK5/p25-dependent phosphorylation of DARPP-32 in mouse striatum slices in vitro. This dual specificity of paullones may turn these compounds into very useful tools for the study and possibly treatment of neurodegenerative and proliferative disorders.

The synthesis of novel quinolino[3,2-d][1]benzazepines and pyrido[3,2-d][1]benzazepines is described. The in vitro antitumor activity of the compounds has been tested in the antitumor screening of the National Cancer Institute (NCI). Several 2,4-diarylpyrido[3,2-d][1]benzazepin-6-ones and -thiones turned out to exhibit considerable cytotoxicity for tumor cells. For studies of SAR within these series, substituents were introduced into the aromatic rings of the parent systems. Compounds from the thiolactam series tended to show higher potency than the corresponding lactams. Prominent compounds with noteworthy activity and remarkable selectivity for renal cancer cell lines are the lactams 10c, 10g, and 10h and the corresponding thiolactams 11c, 11g, and 11h. Methylation of the azepine nitrogen leads to complete loss of activity, whereas annelation of a triazolo ring at the lactam site or transformation of the thiolactam function to a thiolactim ether results in decreased antitumor activity and selectivity. C...

In order to investigate structure-activity relationships in the antiproliferative darpone series, the derivatives 8a and 8b lacking the aryl substituents in either 2- or 4-position were synthesized and evaluated for cancer cell growth inhibition. Since both 8a and 8b were devoid of noteworthy antiproliferative activity, both aryl substituents can be considered as part of the darpone pharmacophore.

The synthesis of some derivatives of the novel heterocyclic ring system pyrido[3,4-d][1]benzazepine is reported. Thus, pyrido[3,4-d][1]benzazepin-7-one 6 was prepared by cyclisation of the Michael adduct 5 with ammonium ferric sulfate. Reaction of 6 with phosphorus pentasulfide gave the thiolactam 7, which after methylation and subsequent reaction with amines furnished the amidines 10.