Palladium-Catalysed Migratory Suzuki-Miyaura Cross-Coupling

The installation of a functional group at a specific site through transition-metal catalysed C-H functionalisation has emerged as a powerful tool for the synthesis of complex structures in a step-economical manner. In this context, the remote functionalisation through migration of a transition-metal catalyst along an alkyl-chain, termed chain-walking, has gained significant momentum during the last decade. The strategy developed by the group of Baudoin relies on palladium-catalysed ligand-controlled chain-walk with enolates or organozinc compounds as nucleophiles, where a site-selective reductive elimination is the terminating step. More recently, the group reported the development of a regioconvergent functionalisation of a regioisomeric mixture of bromoalkanes through a migratory Barbier-Negishi cross-coupling. However, the design of a migratory version of the ubiquitous Suzuki-Miyaura cross-coupling has remained elusive, although various palladium-catalysed migratory cross-coupling reactions were reported. In this optic, we first investigated a one-pot approach for the hydroboration of a regioisomeric mixture of linear alkenes and subsequent terminal-selective palladium-catalysed Suzuki-Miyaura cross-coupling. The developed reaction conditions furnished excellent regioselectivity, but the yields were not satisfactory. We then developed a benzylic-selective palladium-catalysed Suzuki-Miyaura cross-coupling based on the previous observations made. Excellent regioselectivity for the benzylic position of the initial alkene was achieved by the combination of ligand and electrophile. We also demonstrated the regioconvergence from regio- and geometrical isomeric mixture of alkenes, and long-range migration. Additionally, a mechanistic study was also performed. Finally, we also explored the feasibility of combining the previous benzylic-selective migratory cross-coupling with a subsequent C(sp2)-H activation in a cascade process. However, our findings led to the conclusion that the necessary presence of water for the first step is detrimental for the second

Galactosylsphingamides : new α-GalCer analogues to probe the F’-pocket of CD1d

Invariant Natural Killer T-cells (iNKT-cells) are an attractive target for immune response modulation, as upon CD1d-mediated stimulation with KRN7000, a synthetic alpha-galactosylceramide, they produce a vast amount of cytokines. Here we present a synthesis that allows swift modification of the phytosphingosine side chain by amidation of an advanced methyl ester precursor. The resulting KRN7000 derivatives, termed alpha-galactosylsphingamides, were evaluated for their capacity to stimulate iNKT-cells. While introduction of the amide-motif in the phytosphingosine chain is tolerated for CD1d binding and TCR recognition, the studied alpha-galactosylsphingamides showed compromised antigenic properties

Untargeted metabolomics by high resolution mass spectrometry coupled to normal and reversed phase liquid chromatography as a tool to study the in vitro biotransformation of new psychoactive substances

In 2016, several synthetic cathinones were seized by the State Bureau of Criminal Investigation Bavaria in Germany. Due to their previous appearances in other countries their metabolism was already investigated in human urine as well as different in vitro models. These investigations were conducted using ordinary metabolism studies for drugs of abuse by using general knowledge about drug metabolism and visual comparison of mass spectra. The present study aimed to use untargeted metabolomics to support and improve those methods that highly depend on the investigators experience. Incubations were conducted using pooled human liver microsomes (pHLM) and the two cathinones 1-phenyl-2-(1-pyrrolidinyl)-1-butanone and 1-phenyl-2-(1-pyrrolidinyl)-1-heptanone. Samples were analyzed by LC-HRMS/MS using a metabolomics workflow consisting of a reversed phase or normal phase separation followed by electrospray ionization and full scan in positive or negative mode. LC-MS data was afterwards statistically evaluated using principal component analysis, t-distributed stochastic neighborhood embedding, and hierarchical clustering. Significant features were then identified using MS/MS. The workflow revealed 24 significant features after 1-phenyl-2-(1-pyrrolidinyl)-1-butanone and 39 after 1-phenyl-2-(1-pyrrolidinyl)-1-heptanone incubation, consisting of adducts, artifacts, isomers, and metabolites. The applied untargeted metabolomics strategy was able to find almost all of the metabolites that were previously described for 1-phenyl-2-(1-pyrrolidinyl)-1-butanone in literature as well as three additional metabolites. Concerning 1-phenyl-2-(1-pyrrolidinyl)-1-heptanone biotransformation in pHLM, merely four metabolites described in primary human hepatocytes and human urine were not found. This study revealed that untargeted metabolomics workflows are well suited to support biotransformation studies at least of the investigated compounds in pHLM

Reactivating aberrantly hypermethylated p15 gene in leukemic T cells by a phenylhexyl isothiocyanate mediated inter-active mechanism on DNA and chromatin

<p>Abstract</p> <p>Background</p> <p>We have previously demonstrated that phenylhexyl isothiocyanate (PHI), a synthetic isothiocyanate, inhibits histone deacetylases and remodels chromatins to induce growth arrest in HL-60 myeloid leukemia cells in a concentration-dependent manner.</p> <p>Methods</p> <p>To investigate the effect of PHI, a novel histone deacetylases inhibitor (HDACi), on demethylation and activation of transcription of <it>p15 </it>in acute lymphoid leukemia cell line Molt-4, and to further decipher the potential mechanism of demethylation, DNA sequencing and modified methylation specific PCR (MSP) were used to screen <it>p15</it>-M and <it>p15</it>-U mRNA after Molt-4 cells were treated with PHI, 5-Aza and TSA. DNA methyltransferase 1 (DNMT1), 3A (DNMT3A), 3B (DNMT3B) and <it>p15 </it>mRNA were measured by RT-PCR. P15 protein, acetylated histone H3 and histone H4 were detected by Western Blot.</p> <p>Results</p> <p>The gene <it>p15 </it>in Molt-4 cells was hypermethylated and inactive. Hypermethylation of gene <it>p15 </it>was attenuated and <it>p15 </it>gene was activated de novo after 5 days exposure to PHI in a concentration-dependent manner. DNMT1 and DNMT3B were inhibited by PHI (P < 0.05). Alteration of DNMT3A was not significant at those concentrations. Acetylated histone H3 and histone H4 were accumulated markedly after exposure to PHI.</p> <p>Conclusion</p> <p>PHI could induce both DNA demethylation and acetylated H3 and H4 accumulation in Molt-4 cells. Hypermethylation of gene <it>p15 </it>was reversed and <it>p15 </it>transcription could be reactivated de novo by PHI.</p

Toxicometabolomics of cathinone derivatives

Die vorliegende Arbeit untersuchte den Metabolismus der synthetischen Cathinone alpha-PBP, alpha-PVT, alpha-PHP, alpha-PEP, and alpha-POP mit und ohne Metabolomik. Der Metabolismus wurde konventionell untersucht, sowie die Kinetikparameter der beteiligten Cytochrom P450 (CYP) Enzyme bestimmt. Ein ungerichtetes Metabolomik (UM)-Arbeitsschema wurde entwickelt, optimiert und auf Inkubationen mit gepoolten humanen Lebermikrosomen (pHLM), sowie HepaRG Zellen angewandt. Alle untersuchten Cathinone wurden reduziert, bildeten Laktame und wurden an ihren Pyrrolidinringen hydroxyliert. Hauptsächlich am Metabolismus beteiligt waren die polymorph exprimierten CYP2D6, CYP2C9 und CYP2C19. Die Entwicklung des UM Arbeitss- chema zeigte, dass die Parameter der Präprozessierung und die Auflösung des Massenspektrometers wichtig für die Wiederfindung von Analyten sind. Angewandt auf pHLM stellte sich UM als effektiver im Vergleich zu konventionellen Methoden heraus, wobei die Ergebnisse vergleichbar mit primären humanen Hepatozyten waren. Die Untersuchung verschiedener Rekonstitutionsmittelmischungen zeigte, dass eine Zusammensetzung von 30% Methanol und 70% Acetonitril gut geeignet ist, um hohe Feature-Flächen und Gesamtfeaturezahlen zu erhalten. Die Anwendung von UM auf Inkubationen mit HepaRG Zellen führte zur Detektion von unerwarteten Aminosäure-Addukten. Diese Addukte wurden bisher noch nicht in der Literatur beschrieben und können wahrscheinlich auch im Menschen detektiert werden.The presented work investigated the metabolism of the synthetic cathinones alpha-PBP, alpha-PVT, alpha-PHP, alpha-PEP, and alpha-POP with and without metabolomics techniques. The metabolism was elucidated using conventional methods and the kinetic parameters of the involved cytochrome P450 (CYP) enzymes were determined. An untargeted metabolomics (UM) workflow was developed, optimized, and applied to incubations using pooled human liver microsomes (pHLM) and HepaRG cells. All investigated cathinones underwent reduction of the cathinone oxo group, lactam formation and hydroxylation of the pyrrolidine ring. The CYP enzymes involved in the metabolism were polymorphically expressed including CYP2D6, CYP2C9, and CYP2C19. Development of the UM workflow revealed the preprocessing parameters and the resolution of the mass spectrometer as vital parameters effecting the recovery of analytes. The metabolomics approach using pHLM appeared to be more effective than the conventional approach, detecting almost as much metabolites as primary human hepatocytes. The investigation of different reconstitution solvent mixtures showed that a composition of 30% methanol and 70% acetonitrile was well suited to obtain high feature areas and total feature counts. Applying UM to incubations using HepaRG cells led to the detection of unexpected amino acid adducts with the investigated cathinones. These adducts were not described in previous publications and are likely detectable in humans, as well

Comparison of Three Untargeted Data Processing Workflows for Evaluating LC-HRMS Metabolomics Data

The evaluation of liquid chromatography high-resolution mass spectrometry (LC-HRMS) raw data is a crucial step in untargeted metabolomics studies to minimize false positive findings. A variety of commercial or open source software solutions are available for such data processing. This study aims to compare three different data processing workflows (Compound Discoverer 3.1, XCMS Online combined with MetaboAnalyst 4.0, and a manually programmed tool using R) to investigate LC-HRMS data of an untargeted metabolomics study. Simple but highly standardized datasets for evaluation were prepared by incubating pHLM (pooled human liver microsomes) with the synthetic cannabinoid A-CHMINACA. LC-HRMS analysis was performed using normaland reversed-phase chromatography followed by full scan MS in positive and negative mode. MS/MS spectra of significant features were subsequently recorded in a separate run. The outcome of each workflow was evaluated by its number of significant features, peak shape quality, and the results of the multivariate statistics. Compound Discoverer as an all-in-one solution is characterized by its ease of use and seems, therefore, suitable for simple and small metabolomic studies. The two open source solutions allowed extensive customization but particularly, in the case of R, made advanced programming skills necessary. Nevertheless, both provided high flexibility and may be suitable for more complex studies and questions

Impact of the used solvent on the reconstitution efficiency of evaporated biosamples for untargeted metabolomics studies

Introduction Untargeted metabolomics intends to objectively analyze a wide variety of compounds. Their diverse physicochemical properties make it difficult to choose an appropriate reconstitution solvent after sample evaporation without influencing the chromatography or hamper column sorbent integrity. Objectives The study aimed to identify the most appropriate reconstitution solvent for blood plasma samples in terms of feature recovery, four endogenous compounds, and one selected internal standard. Methods We investigated several reconstitution solvent mixtures containing acetonitrile and methanol to resolve human plasma extract and evaluated them concerning the peak areas of tryptophan-d5, glucose, creatinine, palmitic acid, and the phophatidylcholine PC(P-16:0/P-16:0), as well as the total feature count Results Results indicated that acetonitrile containing 30% methanol was best suited to match all tested criteria at least for human blood plasma samples. Conclusion Despite identifying the mixture of acetonitrile and methanol being suitable as solvent for human blood plasma extracts, we recommend to systematically test for an appropriate reconstitution solvent for each analyzed biomatrix

Toxicometabolomics of cathinone derivatives

Die vorliegende Arbeit untersuchte den Metabolismus der synthetischen Cathinone alpha-PBP, alpha-PVT, alpha-PHP, alpha-PEP, and alpha-POP mit und ohne Metabolomik. Der Metabolismus wurde konventionell untersucht, sowie die Kinetikparameter der beteiligten Cytochrom P450 (CYP) Enzyme bestimmt. Ein ungerichtetes Metabolomik (UM)-Arbeitsschema wurde entwickelt, optimiert und auf Inkubationen mit gepoolten humanen Lebermikrosomen (pHLM), sowie HepaRG Zellen angewandt. Alle untersuchten Cathinone wurden reduziert, bildeten Laktame und wurden an ihren Pyrrolidinringen hydroxyliert. Hauptsächlich am Metabolismus beteiligt waren die polymorph exprimierten CYP2D6, CYP2C9 und CYP2C19. Die Entwicklung des UM Arbeitsschema zeigte, dass die Parameter der Präprozessierung und die Auflösung des Massen- spektrometers wichtig für die Wiederfindung von Analyten sind. Angewandt auf pHLM stellte sich UM als effektiver im Vergleich zu konventionellen Methoden heraus, wobei die Ergebnisse vergleichbar mit primären humanen Hepatozyten waren. Die Untersuchung verschiedener Rekonstitutionsmittelmischungen zeigte, dass eine Zusammensetzung von 30% Methanol und 70% Acetonitril gut geeignet ist, um hohe Feature-Flächen und Gesamtfeaturezahlen zu erhalten. Die Anwendung von UM auf Inkubationen mit HepaRG Zellen führte zur Detektion von unerwarteten Aminosäure-Addukten. Diese Addukte wurden bisher noch nicht in der Literatur beschrieben und können wahrscheinlich auch im Menschen detektiert werden.The presented work investigated the metabolism of the synthetic cathinones alpha-PBP, alpha-PVT, alpha-PHP, alpha-PEP, and alpha-POP with and without metabolomics techniques. The metabolism was elucidated using conventional methods and the kinetic parameters of the involved cytochrome P450 (CYP) enzymes were determined. An untargeted metabolomics (UM) workflow was developed, optimized, and applied to incubations using pooled human liver microsomes (pHLM) and HepaRG cells. All investigated cathinones underwent reduction of the cathinone oxo group, lactam formation and hydroxylation of the pyrrolidine ring. The CYP enzymes involved in the metabolism were polymorphically expressed including CYP2D6, CYP2C9, and CYP2C19. Development of the UM workflow revealed the preprocessing parameters and the resolution of the mass spectrometer as vital parameters effecting the recovery of analytes. The metabolomics approach using pHLM appeared to be more effective than the conventional approach, detecting almost as much metabolites as primary human hepatocytes. The investigation of different reconstitution solvent mixtures showed that a composition of 30% methanol and 70% acetonitrile was well suited to obtain high feature areas and total feature counts. Applying UM to incubations using HepaRG cells led to the detection of unexpected amino acid adducts with the investigated cathinones. These adducts were not described in previous publications and are likely detectable in humans, as well

Phenylhexyl isothiocyanate has dual function as histone deacetylase inhibitor and hypomethylating agent and can inhibit myeloma cell growth by targeting critical pathways

Histone deacetylase (HDAC) inhibitors are a new class of chemotherapeutic agents. Our laboratory has recently reported that phenylhexyl isothiocyanate (PHI), a synthetic isothiocyanate, is an inhibitor of HDAC. In this study we examined whether PHI is a hypomethylating agent and its effects on myeloma cells. RPMI8226, a myeloma cell line, was treated with PHI. PHI inhibited the proliferation of the myeloma cells and induced apoptosis in a concentration as low as 0.5 μM. Cell proliferation was reduced to 50% of control with PHI concentration of 0.5 μM. Cell cycle analysis revealed that PHI caused G1-phase arrest of RPMI8226 cells. PHI induced p16 hypomethylation in a concentration- dependent manner. PHI was further shown to induce histone H3 hyperacetylation in a concentration-dependent manner. It was also demonstrated that PHI inhibited IL-6 receptor expression and VEGF production in the RPMI8226 cells, and reactivated p21 expression. It was found that PHI induced apoptosis through disruption of mitochondrial membrane potential. For the first time we show that PHI can induce both p16 hypomethylation and histone H3 hyperacetylation. We conclude that PHI has dual epigenetic effects on p16 hypomethylation and histone hyperacetylation in myeloma cells and targets several critical processes of myeloma proliferation