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Background The determinants of successful humoral immune response to the severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) are of critical importance for the design of effective vaccines and the evaluation of the degree of protective immunity conferred by exposure to the virus. As novel variants emerge, understanding their likelihood of suppression by population antibody repertoires has become increasingly important. Methods In this study, we analyzed the SARS‐CoV‐2 polyclonal antibody response in a large population of clinically well‐characterized patients after mild and severe COVID‐19 using a panel of microarrayed structurally folded and unfolded SARS‐CoV‐2 proteins, as well as sequential peptides, spanning the surface spike protein (S) and the receptor‐binding domain (RBD) of the virus. Results S‐ and RBD‐specific antibody responses were dominated by immunoglobulin G (IgG), mainly IgG1, and directed against structurally folded S and RBD and three distinct peptide epitopes in S2. The virus neutralization activity of patients´ sera was highly correlated with IgG antibodies specific for conformational but not sequential RBD epitopes and their ability to prevent RBD binding to its human receptor angiotensin‐converting enzyme 2 (ACE2). Twenty percent of patients selectively lacked RBD‐specific IgG. Only immunization with folded, but not with unfolded RBD, induced antibodies against conformational epitopes with high virus‐neutralizing activity. Conformational RBD epitopes required for protection do not seem to be altered in the currently emerging virus variants. Conclusion These results are fundamental for estimating the protective activity of antibody responses after natural infection or vaccination and for the design of vaccines, which can induce high levels of SARS‐CoV‐2–neutralizing antibodies conferring sterilizing immunity., IgG response in convalescent COVID‐19 patients is directed to folded but not to unfolded RBD or RBD peptides. IgGs to folded RBD are required for virus neutralization. Twenty percent of convalescent COVID‐19 patients selectively lack RBD‐specific IgG. Only immunization with folded, but not with unfolded RBD, induces antibodies with virus‐neutralizing activity.

In order to investigate whether COVID-19 convalescent patients have developed antibodies that may protect from reinfection, we collected sera from COVID-19 convalescent patients approximately 10 weeks after confirmation of COVID-19 by qRT-PCR (group B, n = 25, 11 females, 14 males, age range: 18-70 years, median age 52.2) and included for control purposes sera from subjects obtained before the COVID-19 pandemic (historic control group P, n = 24, 13 females, 11 males, age range: 18-68 years, median age 43.2). Our findings suggest that a natural SARS-CoV-2 infection, similar to that observed previously for rhinovirus (RV) infections,9 does not induce a protective antibody response inhibiting the virus-receptor interaction in all infected patients and therefore underline the urgent need for the development of a SARS-CoV-2 vaccine. The molecular interaction assays could be useful for identifying subjects having developed protective antibodies and for screening candidate vaccines to induce antibodies that inhibit the RBD-ACE2 interaction once they have been validated.

Background: BM32, a grass pollen allergy vaccine containing four recombinant fusion proteins consisting of hepatitis B-derived PreS and hypoallergenic peptides from the major timothy grass pollen allergens adsorbed on aluminium hydroxide has been shown to be safe and to improve clinical symptoms of grass pollen allergy upon allergen-specific immunotherapy (AIT). We have investigated the immune responses in patients from a two years double-blind, placebo-controlled AIT field trial with BM32. Methods: Blood samples from patients treated with BM32 (n = 27) or placebo (Aluminium hydroxide) (n = 13) were obtained to study the effects of vaccination and natural allergen exposure on allergen-specific antibody, T cell and cytokine responses. Allergen-specific IgE, IgG, IgG1 and IgG4 levels were determined by ImmunoCAP and ELISA, respectively. Allergen-specific lymphocyte proliferation by 3H thymidine incorporation and multiple cytokine responses with a human 17-plex cytokine assay were studied in cultured peripheral blood mononuclear cells (PBMCs). Findings: Two years AIT comprising two courses of 3 pre-seasonal injections of BM32 and a single booster after the first pollen season induced a continuously increasing (year 2 > year 1) allergen-specific IgG4 response without boosting allergen-specific IgE responses. Specific IgG4 responses were accompanied by low stimulation of allergen-specific PBMC responses. Increases of allergen-specific pro-inflammatory cytokine responses were absent. The rise of allergen-specific IgE induced by seasonal grass pollen exposure was partially blunted in BM32-treated patients. Interpretation: AIT with BM32 is characterised by the induction of a non-inflammatory, continuously increasing allergen-specific IgG4 response (year 2 > year1) which may explain that clinical efficacy was higher in year 2 than in year 1. The good safety profile of BM32 may be explained by lack of IgE reactivity and low stimulation of allergen-specific T cell and cytokine responses. Fundings: Grants F4605, F4613 and DK 1248-B13 of the Austrian Science Fund (FWF). Keywords: Allergy, Allergen, Allergen-specific immunotherapy (AIT), Recombinant allergy vaccine, BM32, IgE, IgG subclass, T cell response, Cytokine response

Background SARS‐CoV‐2 has triggered a pandemic that is now claiming many lives. Several studies have investigated cellular immune responses in COVID‐19‐infected patients during disease but little is known regarding a possible protracted impact of COVID‐19 on the adaptive and innate immune system in COVID‐19 convalescent patients. Methods We used multiparametric flow cytometry to analyze whole peripheral blood samples and determined SARS‐CoV‐2‐specific antibody levels against the S‐protein, its RBD‐subunit, and viral nucleocapsid in a cohort of COVID‐19 convalescent patients who had mild disease ~10 weeks after infection (n = 109) and healthy control subjects (n = 98). Furthermore, we correlated immunological changes with clinical and demographic parameters. Results Even ten weeks after disease COVID‐19 convalescent patients had fewer neutrophils, while their cytotoxic CD8+ T cells were activated, reflected as higher HLA‐DR and CD38 expression. Multiparametric regression analyses showed that in COVID‐19‐infected patients both CD3+CD4+ and CD3+CD8+ effector memory cells were higher, while CD25+Foxp3+ T regulatory cells were lower. In addition, both transitional B cell and plasmablast levels were significantly elevated in COVID‐19‐infected patients. Fever (duration, level) correlated with numbers of central memory CD4+ T cells and anti‐S and anti‐RBD, but not anti‐NC antibody levels. Moreover, a “young immunological age” as determined by numbers of CD3+CD45RA+CD62L+CD31+ recent thymic emigrants was associated with a loss of sense of taste and/or smell. Conclusion Acute SARS‐CoV‐2 infection leaves protracted beneficial (ie, activation of T cells) and potentially harmful (ie, reduction of neutrophils) imprints in the cellular immune system in addition to induction of specific antibody responses., Ten weeks after disease, COVID‐19 patients had fewer neutrophils compared to subjects without COVID‐19, while their cytotoxic CD8+ T cells were still activated. In COVID‐19 patients both CD3+CD4+ and CD3+CD8+ effector memory cells, transitional B cells and plasmablast levels were higher, while CD25+Foxp3+ T regulatory cells were lower than in subjects without COVID‐19. Fever duration correlated with higher numbers of central memory CD4+ T cells, anti‐S and anti‐RBD antibody levels, while loss of taste/smell was associated with higher levels of recent thymic emigrants. Abbreviations: COVID‐19, coronavirus disease 2019; ELISA, enzyme‐linked immunosorbent assay; KRECs, kappa‐deleting recombination excision circles; qPCR, quantitative PCR; RBD, receptor‐binding domain of SARS‐CoV2 spike protein; TRECs, T‐cell receptor excision circles; S, spike protein of SARS‐Cov2; SARS‐CoV‐2, severe acute respiratory syndrome coronavirus 2.

Background Chronic hepatitis B virus (HBV) infections are a global health problem. There is a need for therapeutic strategies blocking continuous infection of liver cells. The grass pollen allergy vaccine BM32 containing the preS domain of the large HBV surface protein (LHBs) as immunogenic carrier induced IgG antibodies in human subjects inhibiting HBV infection in vitro. Aim of this study was the quantification, epitope mapping and investigation of HBV genotype cross-reactivity of preS-specific antibodies in subjects treated with different dosage regimens of BM32 Methods Hundred twenty eight grass pollen allergic patients received in a double-blind, placebo-controlled trial five monthly injections of placebo (aluminum hydroxide, n= 34) or different courses of BM32 (2 placebo + 3 BM32, n= 33; 1 placebo + 4 BM32, n= 30; 5 BM32, n= 31). Recombinant Escherichia coli-expressed preS was purified. Overlapping peptides spanning preS and the receptor-binding sites from consensus sequences of genotypes A–H were synthesized and purified. Isotype (IgM, IgG, IgA, IgE) and IgG subclass (IgG1-IgG4) responses to preS and peptides were determined by ELISA at baseline, one and four months after the last injection. IgG1 and IgG4 subclass concentrations specific for preS and the receptor-binding site were measured by quantitative ELISA. Findings Five monthly injections induced the highest levels of preS-specific IgG consisting mainly of IgG1 and IgG4, with a sum of median preS-specific IgG1 and IgG4 concentrations of >135 μg/ml reaching up to 1.8 mg/ml. More than 20% of preS-specific IgG was directed against the receptor-binding site. BM32-induced IgG cross-reacted with the receptor-binding domains from all eight HBV genotypes A-H. Interpretation BM32 induces high levels of IgG1 and IgG4 antibodies against the receptor binding sites of all eight HBV genotypes and hence might be suitable for therapeutic HBV vaccination. Funding This study was supported by the PhD program IAI (KPW01212FW), by Viravaxx AG and by the Danube-ARC funded by the Government of Lower Austria. Rudolf Valenta is a recipient of a Megagrant of the Government of the Russian Federation, grant No 14.W03.31.0024.

Many compounds have been shown to prevent reperfusion injury in various animal models, although to date, translation into clinic has revealed several obstacles. Therefore, the National Heart, Lung, and Blood Institute convened a working group to discuss reasons for such failure. As a result, the concept of adequately powered, blinded, randomized studies for preclinical development of a compound has been urged. We investigated the effects of a fibrin-derived peptide Bbeta(15-42) in acute and chronic rodent models of ischemia-reperfusion at three different study centers (Universities of Dusseldorf and Vienna, TNO Biomedical Research). A total of 187 animals were used, and the peptide was compared with the free radical scavenger Tempol, CD18 antibody, alpha-C5 antibody, and the golden standard, ischemic preconditioning. We show that Bbeta(15-42) robustly and reproducibly reduced infarct size in all models of ischemia-reperfusion. Moreover, the peptide significantly reduced plasma levels of the cytokines interleukin 1beta, tumor necrosis factor alpha, and interleukin 6. In rodents, Bbeta(15-42) inhibits proinflammatory cytokine release and is cardioprotective during ischemia-reperfusion injury.

A novel highly selective single-use IgE-immunoadsorber, IgEnio, has been developed using BM10, a non-anaphylactic fragment of a monoclonal anti-human IgE antibody. In a proof of concept study, IgEnio was used to deplete IgE from the plasma of patients suffering from allergic asthma. In a randomized open-label trial (NCT02096237) 15 highly allergic asthmatics with a total IgE of ≥300U/ml were included. Ten patients were randomly allocated to the treatment group, 5 to a control group. During 16 weeks, every treated patient had a total of 9 IgE-adsorptions: 3 in the 1 st , the 6 th and the 11 th week, respectively. Control visits were scheduled between the weeks of treatment and at the end of the study. Aims of the trial were (1) reduction of total IgE by ≥50% after the last treatment as compared to the beginning of the trial and (2) to demonstrate safety of the new IgE-adsorber. In addition, lung parameters and symptoms were recorded to trace clinical effects. All study participants completed the trial but one, who dropped out for personal reasons before first treatment. Irrespective of individual total-IgE levels (range 308-2518U/ml) IgE was reduced by 79% on average per treatment and by 86.2% (±5.1%) over the entire treatment period. Most adverse events possibly related to the investigational treatment concerned deviations of laboratory parameters and allergic reactions of the skin which are also observed in other immunoadsorption therapies. However, all adverse events were effectively medicated and IgE-adsorptions could be completed in all patients. Using IgEnio, a reliable and safe reduction of total-IgE was achieved, even for extremely elevated IgE-levels. Further studies will focus on clinical efficacy of IgE-adsorption.

Immediate reopening of acutely occluded coronary arteries via primary percutaneous coronary intervention (PCI) is the treatment of choice to salvage the ischemic myocardium in the setting of ST-segment elevation myocardial infarction (STEMI). However, the sudden re-initiation of blood flow achieved with PCI can lead to a local acute inflammatory response with further endothelial and myocardial damage.This phenomenon, described as ‘reperfusion injury’, has been recognized for several decades, yet no pharmacologic intervention has so far succeeded in reducing myocardial damage linked to reperfusion. FX06 is a naturally occurring peptide derived from the neo-N-terminus of fibrin (Bβ15–42). It prevents leukocyte migration through the gap junctions of endothelial cells. Experimental studies have shown that FX06 inhibits the binding of the proinflammatory fibrin E1 fragment to VE-cadherin expressed in the adherence junction. It represents a novel approach to reducing local and systemic inflammation, including myocardial reperfusion injury, in the adherens junction. The present multicenter, double-blind, randomized, placebo-controlled study is designed to test the hypothesis that FX06 injection during and immediately after primary PCI can reduce infarct size in patients with STEMI. The primary outcome measure of efficacy in this study is the degree of myocardial salvage calculated as the difference between the perfusion defect before and after PCI, determined by myocardial perfusion scintigraphy during rest. Further, infarct size at the end of the index hospitalization, as well as at 4 months, will be measured by cardiac magnetic resonance imaging. The present position paper describes the rationale, design and the methods utilized in this trial.

Stickoxid, das Molekul des Jahres 1992, wurde als neuartiger Botenstoff identifiziert, seine Beteiligung an einer Vielzahl von physiologischen Regelvorgangen wurde wahrscheinlich gemacht. Bei einer Anzahl von Erkrankungen spielen Storungen dieser Regelkreise eine Rolle; je nach Art der Storung kann pharmakologisch entweder NO aus exogenen Donor-Molekulen zugefuhrt oder seine endogene Synthase inhibiert werden.

Immediate reopening of acutely occluded coronary arteries via primary percutaneous coronary intervention (PCI) is the treatment of choice to salvage the ischemic myocardium in the setting of ST-segment elevation myocardial infarction (STEMI). However, the sudden re-initiation of blood flow achieved with PCI can lead to a local acute inflammatory response with further endothelial and myocardial damage. This phenomenon, described as 'reperfusion injury', has been recognized for several decades, yet no pharmacologic intervention has so far succeeded in reducing myocardial damage linked to reperfusion. FX06 is a naturally occurring peptide derived from the neo-N-terminus of fibrin (Bbeta(15-42)). It prevents leukocyte migration through the gap junctions of endothelial cells. Experimental studies have shown that FX06 inhibits the binding of the proinflammatory fibrin E1 fragment to VE-cadherin expressed in the adherence junction. It represents a novel approach to reducing local and systemic inflammation, including myocardial reperfusion injury, in the adherens junction. The present multicenter, double-blind, randomized, placebo-controlled study is designed to test the hypothesis that FX06 injection during and immediately after primary PCI can reduce infarct size in patients with STEMI. The primary outcome measure of efficacy in this study is the degree of myocardial salvage calculated as the difference between the perfusion defect before and after PCI, determined by myocardial perfusion scintigraphy during rest. Further, infarct size at the end of the index hospitalization, as well as at 4 months, will be measured by cardiac magnetic resonance imaging. The present position paper describes the rationale, design and the methods utilized in this trial.

The antiischemic effect of pirsidomine (CAS 936 (3-(cis-2,6-dimethylpiperidino)-N-(4-methoxybenzoyl))-sydnon imine), a new nitric oxide donor, was investigated in a model of myocardial infarction in the dog. Dogs were anaesthetised, thoracotomized, and the left descending coronary artery was occluded for 6 h. Pirsidomine was given intraduodenally (i.d.) at the dose of 1.0 mg/kg to 11 dogs 30 min prior to coronary occlusion. Eleven dogs received the solvent i.d. and served as controls. Pirsidomine administration completely prevented the increase in left ventricular end-diastolic pressure and pulmonary artery pressure induced by the coronary occlusion and resulted in a marked decrease in systolic and diastolic blood pressure, cardiac output, left ventricular contractility, left ventricular work and left ventricular oxygen consumption. Additionally, pirsidomine completely prevented the occlusion-induced increase in flow in the non-occluded circumflex coronary artery. Regional blood flow measurements (with radioactive microspheres) revealed that pirsidomine induced a significant reduction in blood flow in the non-ischemic areas (both epi- and endocardial) but in the course of the ischemia, significantly increased flow in the ischemic epicardial areas. Infarct-size (triphenyltetrazolium chloride technique) in control dogs was 45% of the area at risk, but only 26% (P < 0.05) in pirsidomine-treated dogs. Thus, pirsidomine had a marked antiischemic effect in this model. This was probably due to the hemodynamic unloading of the heart as well as to redistribution of blood from the non-ischemic to the ischemic areas of the myocardium.

Nitric oxide is produced by the NO synthase, which catalyses the conversion of arginine to citrulline and NO using tetrahydrobiopterin as an essential cofactor. N-Acetylserotonin, an inhibitor of the tetrahydrobiopterin biosynthesis, given 30 min before bacterial lipopolysaccharide to anesthetized rats, inhibited both the decrease in blood pressure and the increase in nitrite plasma levels induced by lipopolysaccharide. Thus, during endotoxemia the availability of tetrahydrobiopterin appears to be essential for the activity of NO synthase.

Based on the concept of transition-state analogs, a series of nonpeptide renin inhibitors with the new (2S,3R,4S)-2-amino-1-cyclohexyl-3,4-dihydroxy-6-(2-pyridyl)hexane moiety at the C-terminal functionality were synthesized and evaluated for inhibition of renin both in vitro and in vivo. All compounds exhibited potencies in the nanomolar or even subnanomolar range when tested versus human renin in vitro. Selected inhibitors were evaluated in anesthetized, sodium-depleted rhesus monkeys and produced a marked reduction in mean arterial blood pressure (MAP) upon intraduodenal administration of a dose of 2 mg/kg. Compound 38 (S 2864) containing an amino piperidyl succinic acid derived N-terminal is the most promising member in this series. 38 inhibited human renin with an IC50 of 0.38 nM, did not affect other human aspartic proteinases, and decreased mean arterial blood pressure significantly by 27% with a duration of action of 90 min after administration of 2 mg/kg id in anesthetized, sodium-depleted rhesus monkeys.

FX06 (fibrin derived peptide Bβ15-42) is a promising new drug candidate indicated for the prevention of reperfusion injury in patients undergoing primary percutaneous intervention (PCI) for acute myocardial infarction (AMI). This novel peptide has been shown to prevent myocardial reperfusion injury in a number of acute and chronic animal models for this disease when given as a single bolus injection at the time of reperfusion. FX06 has a novel mechanism of action: it is a competitive inhibitor of the binding of fibrin E1 fragments to vascular endothelial (VE)-cadherin. Through this inhibition, it potently blocks the transmigration of inflammatory leukocytes through the endothelial barrier and prevents the downstream release of tissue-damaging mediators. FX06 has proven safe in acute and subchronic toxicological studies and recently entered clinical development.

1 The present investigation compares the effects of intravenous infusion of the NO synthase inhibitor NG-monomethyl-l-arginine (l-NMMA) with that of an inhalation with NO gas in a porcine model of endotoxin (lipopolysaccharide, LPS) shock. In addition, the effects of the combination of these two treatments were also investigated. 2 Male pigs were anaesthetized and instrumented for the measurement of haemodynamic parameters. Blood samples were withdrawn at different time intervals for determination of blood gases, pH, and plasma levels of nitrite/nitrate and tumour necrosis factor. 3 Endotoxin infusion (15 μg kg−1 h−1 for 3 h) caused a progressive fall in mean arterial blood pressure (MABP) and cardiac output (CO) and a biphasic increase in mean pulmonary arterial pressure (MPAP) and pulmonary vascular resistence (PVR). A continuous infusion of l-NMMA (0.1 mg kg−1 min−1) significantly attenuated the fall in MABP, but did not affect MPAP, CO and PVR. NO-inhalation (50 p.p.m.) did not affect MABP, but significantly blunted the biphasic increase in MPAP and PVR and significantly delayed the fall in CO. The combination of l-NMMA infusion (0.1 mg kg−1 min−1) with NO-inhalation (50 p.p.m.) completely prevented the fall in MABP, significantly improved CO, and attenuated the biphasic increase in MPAP and PVR. 4 Endotoxin also caused a decline in Pao2 and a rise of Paco2. Infusion of l-NMMA neither affected the fall in Pao2 nor the increase in Paco2. In contrast, inhalation with NO gas alone as well as the combined administration of l-NMMA infusion and NO-inhalation completely prevented the fall in Pao2 and significantly protected against the increase in Paco2. 5 Infusion of endotoxin for 180 min resulted in a mortality of 58%, which was not affected by l-NMMA (63%). In contrast, treatment of LPS-animals with either NO-inhalation alone or NO-inhalation plus l-NMMA completely prevented mortality. 6 This investigation demonstrates that treatment with NO-inhalation, in order to prevent the dramatic increase in MPAP, PVR and the alterations in peripheral blood gases combined with systemic l-NMMA to improve systemic MABP and thus organ perfusion, may be a new therapeutic regimen in the treatment of septic shock.

α,β-Doubly deprotonated nitroalkanes: Super-enamines? At temperatures between −90° and −78° both the α and β-proton of 1-aryl-2-nitro-ethanes (1) are abstracted by n-butyllithium to give the dilithio derivatives of 3. These turn out to be excellent nucleophiles combining with alkyl halides, aldehydes, ketones, and ω-nitro styrenes at the β-nitro carbon atom to give products of type 2. It is shown that 2-nitro-propane undergoes the same double deprotonation and can be coupled with benzaldehyde at one of the β-nitro carbon atoms to yield 4. It is proposed to consider the new reagents as super-enamines 3c.

Methyl 3-nitropropanoate can be doubly deprotonated with LDA, both in the α-nitro and in the α-carbonyl position (→ 1). It is shown that the necessary cosolvent HMPT can be replaced by the cyclic urea DMPU, with almost equal results. The new reagent 1 is alkylated and hydroxyalkylated by alkyl halides and aldehydes, respectively, at the 2-position exclusively (see 2, 4, 6, 7). A double alkylation to methyl α,α-dialkyl-β-nitropropanoate 5 is possible. Elimination of HNO2 with DBN or even better with DBU in THF furnishes methyl α-methylenealkanoates 9 and methyl β-hydroxy-α-methylenealkanoates 10.

Among a number of 49 metal sulfur complexes investigated, zinc dithiolenes enable the most efficient catalytic hydrogen production from water upon UV photolysis in the presence of mild reducing agents such as tetrahydrofuran, 2,5-dihydrofuran, and sodium formate. Water cleavage is substantiated by the use of deuterated solvent systems. In the case of metal dithiolenes, the reaction rates depend on the nature of the ligand, the central metal, and on the pH value. Methanol is an efficient reducing agent only in alkaline solution. The metal dithiolenes are transformed into catalysts at the early stages of reaction. These catalytic systems are stable over several days, and maximum turnover numbers of 2000–4000 are obtained. Electron scavengers such as N 2 O and SF 6 inhibit the hydrogen formation.

A direct central nervous system (CNS)-related component of the cardiovascular actions of converting enzyme (CE) inhibitors will be governed by the ability of these drugs to gain access to the brain. We investigated the inhibitory effect of the two CE inhibitors ramipril and Hoe 288 on CE activity in different brain regions and in the cerebrospinal fluid of rats. One-week oral gavage treatment with ramipril (10 mg/kg/day) and Hoe 288 (10 mg/kg/day) resulted in a marked inhibition of CE activity in the brain cortex (90 and 91%, respectively), the hypothalamus (78 and 82%, respectively), and in the brainstem (67 and 66%, respectively). The complete blockade of plasma CE activity was paralleled by a 84% inhibition of CE activity in cerebrospinal fluid. Both CE inhibitors failed significantly to inhibit CE activity in the striatum. Our results demonstrate that the CE inhibitors Hoe 288 and ramipril were able to pass the blood-brain barrier (BBB) to inhibit central CE activity. The penetration of CE inhibitors into the CNS appears to depend on the lipophilicity of the drugs and on the mode of drug application. The possibility that an inhibition of CE activity in circumventricular organs outside the BBB such as the subfornical organ and the organum vasculosum of the lamina terminalis may be sufficient to explain the central effects of orally applied CE inhibitors is discussed.

β-Arylnitroethane 4 konnen bei −80 bis −90°C in HMPTA-haltigem THF mit zwei Equivalenten n-Butyllithium zu den Dilithiumderivaten 6 doppelt deprotoniert werden, die man als Reduktionsprodukte (s. 3 und 16b) von ω-Nitrostyrolen 2 auffassen kann. Sie reagieren mit Alkylhalogeniden ( 7), Aldehyden und Ketonen ( 8), α,β-ungesattigten Ketonen ( Michael-Addukte 13 und 14) und ω-Nitrostyrolen ( 15) am benzylischen C-Atom. Saurechlorid und Trimethylchlorsilan greifen wahrscheinlich am Sauerstoff an (s. Zwischenprodukte 17–19). Als mit den chemischen Befunden am besten vereinbare Schreibweise fur die neuartigen Reagentien schlagen wir die von Super-enaminen16b vor; in Tab. 1 ist ihre Ladungsaffinitat mit der von Nitroethanen (den Vorlaufern) und von Nitroethylenen (den oxidierten Formen, s. 2 3) verglichen und gezeigt, das sie umgepolte Nitroolefine oder Homonitronate sind. Die Gegenuberstellung in Tab. 2 demonstriert. Das die Dilithiumverbindungen 6 als Synthesebausteine fur Amine und Carbonylverbindungen normale Reaktivitat haben („Doppelumpolung”). Double Deprotonation of 2-Aryl-1-nitroethanes. — Umpolung of the Nitroolefin Reactivity β-Arylnitroethanes 4 are doubly deprotonated to the dilithio derivatives 6 when treated with two equivalents of n-butyllithium in HMPTA-containing THF at −80 to −90°C. The reagents (6) may be considered as the products (see 3 and 16b) of reduction of ω-nitrostyrenes 2, They react with haloalkanes ( 7), aldehydes and ketones ( 8), α,β-unsaturated ketones ( Michael adducts 13 and 14), and ω-nitrostyrenes ( 15) in the benzylic position. With acyl chloride and chloro-trimethylsilane attack occurs probably on an oxygen of 6 (see intermediates 17–19). A representation of the novel reagents, best compatible with the chemical results, is that of super-enamines16b; their charge affinity is compared with that of nitroethanes (the precursors) and that of nitroethylenes (the oxidized forms, see 2 3), and it is shown in table 1 that they are nitroolefins with reactivity umpolung or homonitronates. The comparison made in table 2 demonstrates that the dilithio derivatives 6 are synthetically equivalent with enamines and enolates of normal reactivity (“double umpolung”).

Several neuropeptides have recently been found to be involved in motivational, memory, and learning processes. Therefore, we tested the hypothesis that the peptidase (EC 3.4.15.1) inhibitor Hoe 288 modulated cognitive functions. Hoe 288 exhibited nootropic effects in uphill avoidance in mice (30 mg/kg orally administered [p.o.]). Scopolamineinduced amnesia in mice and rats was antagonized after subchronic administration for two or three days (1–10 mg/kg p.o. or intraperitoneally [i.p.]) in step-through avoidance and an eight-arm-radial maze, respectively. The resulting bell-shaped dose-response curves had a maximum effect at 3 mg/kg. To elucidate the underlying neurochemical effects, ex vivo experiments in rats were performed: acute or repeated administration of Hoe 288 (0.03 – 3 mg/kg i.p.) induced a significant decrease of acetylcholine (ACh) content in certain brain areas that lasted up to 6 hr after treatment. The reduced brain ACh content was most likely due to an enhanced ACh release, as coadministration of the choline uptake inhibitor hemicholinium-3 (HC-3) and Hoe 288 potentiated the decrease of ACh induced by HC-3. Choline acetyltransferase activity in brain increased after acute and subchronic administration of the peptidase inhibitor. Receptor-mediated effects of ACh were estimated indirectly by its stimulation of guanylate cyclase. Hoe 288 increased cGMP content in a comparable dose range (0.3 – 1 mg/kg i.p.). The converting enzyme inhibitor Hoe 288 was shown to influence acquisition and retention of information in rodents, and the effects on ACh metabolism are in concordance with results obtained with potential nootropics in animal experiments.

Derivate von 1,3- und 1,4-Dicarbonylverbindungen (3, 5) entstehen durch Umsetzung der Michael-Addukte von thioacetal-maskierten Acyl-Anionen bzw. Enolaten an Nitroolefine (2, 4) nach dem Prinzip der Nef-Reaktion. Reduktion derselben Addukte (6, 8) mit Lithiumaluminiumhydrid in Tetrahydrofuran/Dioxan fuhrt zu Aminen (7, 9), aus denen leicht Indole (z. B. Bufotenin, 10) oder Isochinoline (z. B. 4-Dithianyl-Derivate 15, 17) nach bekannten Verfahren hergestellt werden konnen. Auch Zugangswege zu 1-Dithianylisochinolinen (z. B. 25) werden beschrieben. Alle diese Transformationen sind in praparativ nutzlichem Masstab und in guten Ausbeuten durchfuhrbar. – Der Einflus von Dithianylgruppen auf die NMR-Verschiebung peri-standiger aromatischer Wasserstoffe im Isochinolin wird angegeben. Conversions of Michael Adducts of Nitroolefins Preparation of Carbonyl Compounds, 1,4-Diaminobutanes, Indoles, and Isoquinolines Derivatives of 1,3- and 1,4-dicarbonyl-systems (3, 5) are prepared from the Michael adducts of masked acyl anion equivalents or enolates, respectively, to nitroolefins (2, 4) using Nef-type reactions. The same adducts (6, 8) are reduced with lithium aluminium hydride in THF/dioxane to give amines (7, 9) which are convenient precursors to indoles (cf. bufotenin, 10) or isoquinolines (cf. 4-dithianyl derivatives 15, 17) if subjected to standard reactions such as the Bischler-Napieralski-, the Pictet-Spengler-, and oxidative indole-ring closures. An access to 1-dithianylisoquinolines (e. g. 25) is also described. All these transformations can be done on a preparative scale and in good to excellent yields. The effects of 1- and 4-dithianyl substituents on the n.m.r. spectral shifts of peri-hydrogens at C-5 and C-8 in di- and tetrahydroisoquinolines are discussed.

Derivate von 1,3- und 1,4-Dicarbonylverbindungen (3, 5) entstehen durch Umsetzung der Michael-Addukte von thioacetal-maskierten Acyl-Anionen bzw. Enolaten an Nitroolefine (2, 4) nach dem Prinzip der Nef-Reaktion. Reduktion derselben Addukte (6, 8) mit Lithiumaluminiumhydrid in Tetrahydrofuran/Dioxan fuhrt zu Aminen (7, 9), aus denen leicht Indole (z. B. Bufotenin, 10) oder Isochinoline (z. B. 4-Dithianyl-Derivate 15, 17) nach bekannten Verfahren hergestellt werden konnen. Auch Zugangswege zu 1-Dithianylisochinolinen (z. B. 25) werden beschrieben. Alle diese Transformationen sind in praparativ nutzlichem Masstab und in guten Ausbeuten durchfuhrbar. – Der Einflus von Dithianylgruppen auf die NMR-Verschiebung peri-standiger aromatischer Wasserstoffe im Isochinolin wird angegeben. Conversions of Michael Adducts of Nitroolefins Preparation of Carbonyl Compounds, 1,4-Diaminobutanes, Indoles, and Isoquinolines Derivatives of 1,3- and 1,4-dicarbonyl-systems (3, 5) are prepared from the Michael adducts of masked acyl anion equivalents or enolates, respectively, to nitroolefins (2, 4) using Nef-type reactions. The same adducts (6, 8) are reduced with lithium aluminium hydride in THF/dioxane to give amines (7, 9) which are convenient precursors to indoles (cf. bufotenin, 10) or isoquinolines (cf. 4-dithianyl derivatives 15, 17) if subjected to standard reactions such as the Bischler-Napieralski-, the Pictet-Spengler-, and oxidative indole-ring closures. An access to 1-dithianylisoquinolines (e. g. 25) is also described. All these transformations can be done on a preparative scale and in good to excellent yields. The effects of 1- and 4-dithianyl substituents on the n.m.r. spectral shifts of peri-hydrogens at C-5 and C-8 in di- and tetrahydroisoquinolines are discussed.

The pharmacological and binding properties of the novel enantiomerically pure benzothiazinone (R)-(+)-3,4-dihydro-2-isopropyl-4-methyl-2-[2-[4-[4-[2-(3,4,5-tri- methoxyphenyl)-ethyl]-piperazinyl]-butoxyl-phenyl]-2H-1,4- benzothiazine-3-one dihydrochloride (HOE 166), are described. HOE 166 stereoselectively inhibited KCl-but not noradrenaline-induced contractions of guinea-pig pulmonary arteries, rabbit aorta, rat mesenteric artery preparations and k-strophantin-induced enhancement of guinea-pig papillary muscle contraction in a dose-dependent manner. KCl-induced smooth muscle contraction was inhibited by HOE 166 with IC50-values of approximately 70 nM (5-11 times less potent than nifedipine, 2-16 times more potent than verapamil), the respective S-(-)-enantiomer being approximately 10-fold less potent. HOE 166 decreased the upstroke velocity of the slow action potential in partially depolarized guinea-pig papillary muscle at similar concentrations than nifedipine. To investigate possible interactions with the calcium channel, HOE 166 and its S-(-)-enantiomer were characterized by radioligand binding studies in heart, brain and skeletal muscle transverse-tubule membranes. HOE 166 was a 4-15 times more potent inhibitor of reversible (+)-[3H]PN200-110, (-)-[3H]desmethoxyverapamil and d-cis [3H]diltiazem binding compared to its pharmacologically less active (S)-(-)-enantiomer, with IC50 values in the low nanomolar range. Extensive equilibrium and kinetic studies suggest that HOE 166 exerts its Ca2+-antagonistic effect by binding to a Ca2+-channel-associated drug receptor which is distinct from the 1,4-dihydropyridine, phenylalkylamine or benzothiazepine-selective domain. This HOE 166-selective site is, however, allosterically linked to the other sites of the Ca2+ antagonist receptor complex. We conclude that HOE 166 is a novel calcium antagonist.