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Objectives: Urinary steroid profiling after hydrolysis of conjugates is an emerging tool to differentiate aggressive adrenocortical carcinomas (ACC) from benign adrenocortical adenomas (ACA). However, the shortcomings of deconjugation are the lack of standardized and fully validated hydrolysis protocols and the loss of information about the originally conjugated form of the steroids. This study aimed to evaluate the quality of the deconjugation process and investigate novel diagnostic biomarkers in urine without enzymatic hydrolysis., Methods: 24 h urine samples from 40 patients with ACC and 40 patients with ACA were analyzed by untargeted metabolomics using liquid chromatography-high-resolution mass spectrometry both unmodified and after hydrolysis with arylsulfatase/glucuronidase from Helix pomatia. Both approaches were compared regarding the differentiation of ACC vs. ACA via ROC analyses and to evaluate the hydrolyzation efficiency of steroid conjugates., Results: Steroid glucuronides were fully deconjugated, while some disulfates and all monosulfates were still largely detectable after enzymatic hydrolysis, suggesting incomplete and variable deconjugation. In unhydrolyzed urine, steroid monosulfates showed the best differentiation between ACC and ACA (highest AUC=0.983 for C 21 H 32 O 6 S, followed by its isomer and two isomers with the molecular formula C 21 H 32 O 7 S). Moreover, several disulfates were highly abundant and increased in ACC compared to ACA., Conclusions: This work highlights the limitations of hydrolyzing steroid conjugates before analysis and shows a possible superiority of a direct analysis approach compared to a hydrolysis approach from a methodological point of view and regarding diagnostic accuracy. Several steroid conjugates were found as promising diagnostic biomarkers for differentiation between ACC and ACA., (© 2024 Walter de Gruyter GmbH, Berlin/Boston.)

The inhibition of renal transport proteins organic cation transporter 2 (OCT2), multidrug and toxin extrusion proteins (MATE1, MATE2-K), and organic anion transporters (OAT1, OAT3) causes clinically relevant drug-drug interactions (DDI). Endogenous biomarkers could be used to improve risk prediction of such renal DDIs. While a number of biomarkers for renal DDIs have been described so far, multiple criteria for valid biomarkers have frequently not been investigated, for example, specificity, metabolism, or food effects. Therefore, there is a need for novel biomarkers of renal DDIs. Here, we investigated the global metabolomic effects following the administration of two classical inhibitors of renal transport proteins [cimetidine (OCT2/MATEs), probenecid (OATs)] in human plasma and urine of healthy volunteers. Additionally, we investigated metabolomic effects of two inhibitors of other transporters [verapamil (P-glycoprotein), rifampin (organic anion transporting polypeptides)] as controls. This analysis shows that both cimetidine and probenecid affect compounds involved in caffeine metabolism, carnitines, and sulfates. Hierarchical cluster analysis of the effects of all four inhibitors on endogenous compounds identified multiple promising new sensitive and specific biomarker candidates for OCT2/MATE- or OAT-mediated DDIs. For OCT2/MATEs, 5-amino valeric acid betaine (median log 2 -fold change of estimated renal elimination: -3.62) presented itself as a promising candidate. For OATs, estimated renal elimination of 7-methyluric acid and cinnamoylglycine (median log 2 -fold changes -3.10 and -1.92, respectively) was both sensitive and specific. This study provides comprehensive information on metabolomic effects of transport protein inhibition in humans and identifies putative new sensitive and specific biomarkers for renal transporter-mediated DDIs., (© 2024 The Author(s). Clinical Pharmacology & Therapeutics published by Wiley Periodicals LLC on behalf of American Society for Clinical Pharmacology and Therapeutics.)

The determination of ligand-receptor binding affinities plays a key role in the development process of pharmaceuticals. While the classical radiochemical binding assay uses radioligands, fluorescence-based binding assays require fluorescent probes. Usually, radio- and fluorescence-labeled ligands are dissimilar in terms of structure and bioactivity, and can be used in either radiochemical or fluorescence-based assays. Aiming for a close comparison of both assay types, we synthesized tritiated fluorescent neurotensin receptor ligands ([ 3 H] 13 , [ 3 H] 18 ) and their nontritiated analogues ( 13 , 18 ). The labeled probes were studied in radiochemical and fluorescence-based (high-content imaging, flow cytometry, fluorescence anisotropy) binding assays. Equilibrium saturation binding yielded well-comparable ligand-receptor affinities, indicating that all these setups can be used for the screening of new drugs. In contrast, discrepancies were found in the kinetic behavior of the probes, which can be attributed to technical differences of the methods and require further studies with respect to the elucidation of the underlying mechanisms.

Myocardin-related transcription factor A (MRTF-A) is a coactivator of serum response factor (SRF), which regulates the expression of genes involved in cell proliferation, migration, and differentiation and has been implicated in hepatocellular carcinoma (HCC) progression. We recently established inhibition of the transcriptional activity of MRTF-A by NS8593 as a novel therapeutic approach for HCC therapy. NS8593 is a negative gating modulator of the transient receptor potential cation channel TRPM7. In this report, we identify an aminobenzimidazole that is highly potent in inhibiting TRPM7 and its interaction with RhoA, leading to decreased SRF transcriptional activity and enhanced nuclear export of MRTF-A, as determined by fluorescence loss in photobleaching (FLIP). This resulted in reduced expression of the MRTF/SRF target genes transforming growth factor β1 (TGF-β1) and tetraspanin 5 (TSPAN5), senescence induction, and growth arrest in HCC cells. Replacement of the tetraline core by a 3-aminophenyl substructure yielded inhibitor 10 with higher potency than inhibitor 5, and further structural modifications yielded highly potent inhibitors of SRF activity, 14 and 16 . Both compounds were capable of inhibiting cell proliferation and inducing senescence in HCC cells with improved efficacy compared to NS8593. These inhibitors represent valuable tools for understanding the molecular basis of drug development targeting TRPM7 and MRTFs., Competing Interests: The authors declare no competing interests., (© 2024 The Author(s).)

The neuropeptide Y (NPY) Y 4 receptor (Y 4 R), a member of the family of NPY receptors, is physiologically activated by the linear 36-amino acid peptide pancreatic polypeptide (PP). The Y 4 R is involved in the regulation of various biological processes, most importantly pancreatic secretion, gastrointestinal motility, and regulation of food intake. So far, Y 4 R binding affinities have been mostly studied in radiochemical binding assays. Except for a few fluorescently labeled PP derivatives, fluorescence-tagged Y 4 R ligands with high affinity have not been reported. Here, we introduce differently fluorescence-labeled (Sulfo-Cy5, Cy3B, Py-1, Py-5) Y 4 R ligands derived from recently reported cyclic hexapeptides showing picomolar Y 4 R binding affinity. With p K i values of 9.22-9.71 (radioligand competition binding assay), all fluorescent ligands ( 16 - 19 ) showed excellent Y 4 R affinity. Y 4 R saturation binding, binding kinetics, and competition binding with reference ligands were studied using different fluorescence-based methods: flow cytometry (Sulfo-Cy5, Cy3B, and Py-1 label), fluorescence anisotropy (Cy3B label), and NanoBRET (Cy3B label) binding assays. These experiments confirmed the high binding affinity to Y 4 R (equilibrium p K d : 9.02-9.9) and proved the applicability of the probes for fluorescence-based Y 4 R competition binding studies and imaging techniques such as single-receptor molecule tracking., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)

Derivatives of the rhodamine-based dye 5-TAMRA (5-carboxy-tetramethylrhodamine) and the indocarbocyanine-type Cy3B (cyclized derivative of the cyanine dye Cy3), both representing important fluorophores frequently used for the labeling of biomolecules (proteins, nucleic acids) and bioactive compounds, such as receptor ligands, were photophysically investigated in aqueous solution, i.e., in neat phosphate-buffered saline (PBS) and in PBS supplemented with 1 wt % bovine serum albumin (BSA). The dyes exhibit comparable absorption (λ abs,max : 550-569 nm) and emission wavelengths (λ em,max : 580-582 nm), and similar S 1 lifetimes (2.27-2.75 ns), and their excited state deactivation proceeds mainly via the lowest excited singlet state (triplet quantum yield ca. 1%). However, the probes show marked differences with respect to their fluorescence quantum yield and photostability. While 5-TAMRA shows a lower quantum yield (37-39%) than the Cy3B derivative (ca. 57%), its photostability is considerably higher compared to Cy3B. Generally, the impact of the protein on the photophysics is low. However, on prolonged illumination, both fluorescent dyes undergo a photocatalytic reaction with tryptophan residues of BSA mediated by sensitized singlet oxygen resulting in a tryptophan photoproduct with an absorption maximum around 330 nm. The overall results of this work will assist in choosing the right dye for the labeling of bioactive compounds, and the study demonstrates that experiments performed with 5-TAMRA or Cy3B-labeled compounds in a biological environment may be influenced by photochemical modification of experimentally relevant proteins at aromatic amino acid residues.

Endogenous biomarkers are discussed as tools for detection of drug-drug interactions (DDIs) mediated by renal transport proteins, such as organic cation transporter 2 (OCT2), multidrug and toxin extrusion proteins (MATE1 and MATE2-K) and organic anion transporters (OAT1 and OAT3). Whereas sensitivity of some endogenous biomarkers against at least one clinical transporter inhibitor has frequently been shown, intra-study comparisons of the extent of effects of inhibitors on different biomarkers are frequently lacking. Moreover, in vivo specificity of such discussed biomarkers has frequently not been studied. We therefore investigated changes of 10 previously described putative biomarkers for inhibition of OCT2/MATEs, as well as 15 previously described putative biomarkers for OATs in human plasma and urine samples of healthy volunteers in response to treatment with 4 inhibitors of transport proteins [verapamil (P-glycoprotein), rifampin (organic anion transporting polypeptides), cimetidine (OCT2/MATEs), and probenecid (OATs)]. Two of the putative biomarkers had been suggested for both OCT2/MATEs and OATs. All substances were unequivocally identified in an untargeted metabolomics assay. The OCT2/MATE biomarkers choline and trimethylamine N-oxide were both sensitive and specific (median log2-fold changes -1.18 in estimated renal elimination and -0.85 in urinary excretion, respectively). For renal OATs, indoleacetyl glutamine and indoleacetic acid (median log2-fold changes -3.77 and -2.85 in estimated renal elimination, respectively) were the candidates for sensitive and specific biomarkers with the most extensive change, followed by taurine, indolelactic acid, and hypoxanthine. This comprehensive study adds further knowledge on sensitivity and specificity of 23 previously described biomarkers of renal OCT2/MATE- and OAT-mediated DDIs., (© 2023 The Authors. Clinical Pharmacology & Therapeutics published by Wiley Periodicals LLC on behalf of American Society for Clinical Pharmacology and Therapeutics.)

Multidrug-resistant bacteria pose a major threat to global health, even as newly introduced antibiotics continue to lose their therapeutic value. Against this background, deeper insights into bacterial interaction with antibiotic drugs are urgently required, whereas fluorescently labeled drug conjugates can serve as highly valuable tools. Herein, the preparation and biological evaluation of 13 new fluorescent antibiotic-Cy5 dye conjugates is described, in which the tuning of the polarity of the Cy5 dye proved to be a key element to achieve highly favorable properties for various fields of application., (© 2023 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH.)

Despite their long history and their synthetic potential underlined by various recent advances, radical thiol-yne coupling reactions have so far only rarely been exploited for the functionalization of biomolecules, and no examples yet exist for their application in live cells - although natural thiols show widespread occurrence therein. By taking advantage of the particular cellular conditions of mitochondria in cancer cells, we have demonstrated that radical thiol-yne coupling represents a powerful reaction principle for the selective targeting of these organelles. Within our studies, fluorescently labeled reactive alkyne probes were investigated, for which the fluorescent moiety was chosen to enable both mitochondria accumulation as well as highly sensitive detection. After preliminary studies under cell-free conditions, the most promising alkyne-dye conjugates were evaluated in various cellular experiments comprising analysis by flow cytometry and microscopy. All in all, these results pave the way for improved future therapeutic strategies relying on live-cell compatibility and selectivity among cellular compartments., (© 2023 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH.)

Optical storage and photon quantification systems based on sensitive photoreactions have numerous applications. Herein, we report a highly efficient photocatalytic reaction, in which ruthenium photoredox catalysis is combined with a 1,2-dioxetane from which chemiluminescence can be triggered. In this system, blue light irradiation as optical input enables a defined inverse correlation with base-triggered, blue light emission as optical output. Comparison of readout by 1 H NMR and chemiluminescence, relative to previous optical input, underlines the reliability and usefulness of the ruthenium-dioxetane system for optical storage, sensing and ruthenium detection., (© 2023 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

By using active pharmaceutical ingredients (APIs) previously recovered from expired drugs, it is shown that Selectfluor can act as a reagent for operationally simple late-stage fluorination and chlorination of electron-rich arenes under mild reaction conditions. As shown in mechanistic experiments, aromatic fluorination thereby competes with chlorine-for-fluorine exchange on Selectfluor and subsequent aromatic chlorination, whereat the chloride ions may either be provided by the hydrochloride salt of the respective API or by triethylammonium chloride. Biological testing of the fluorinated or chlorinated APIs at adrenergic, dopaminergic, muscarinergic, opioid or serotoninergic receptors demonstrated that improved binding affinities can be achieved via this straightforward strategy., (© 2023 The Authors. ChemMedChem published by Wiley-VCH GmbH.)

The high affinity dopamine D 4 receptor ligand APH199 and derivatives thereof exhibit bias toward the G i signaling pathway over β-arrestin recruitment compared to quinpirole. Based on APH199, two novel groups of D 4 subtype selective ligands were designed and evaluated, in which the original benzyl phenylsemicarbazide substructure was replaced by either a biphenylmethyl urea or a biphenyl urea moiety. Functional assays revealed a range of different bias profiles among the newly synthesized compounds, namely, with regard to efficacy, potency, and GRK2 dependency, in which bias factors range from 1 to over 300 and activation from 15% to over 98% compared to quinpirole. These observations demonstrate that within bias, an even more precise tuning toward a particular profile is possible, which─in a general sense─could become an important aspect in future drug development. Docking studies enabled further insight into the role of the ECL2 and the EPB in the emergence of bias, thereby taking advantage of the diversity of functionally selective D 4 agonists now available.

Functionalizations of arenes and alkenes via additive-free radical reactions using highly photosensitive, fluorescein-derived diazonium salts are described. The particular properties of the diazonium salts enable unique Meerwein-type carbohydroxylations of non-activated alkenes, which can be rationalized by a reaction mechanism involving forth and back electron transfer from and to the xanthene subunit of the fluorescein moiety.

Through the linkage of two muscarinergic M 3 receptor ligands to fluorescent tetramethylrhodamine- and cyanine-5-type dyes, two novel tool compounds, OFH5503 and OFH611, have been developed. Based on the suitable binding properties and kinetics related to the M 3 subtype, both ligand-dye conjugates were found to be useful tools to determine binding affinities via flow cytometric measurements. In addition, confocal microscopy underlined the comparably low unspecific binding and the applicability for studying M 3 receptor expression in cells. Along with the proven usefulness regarding studies on the M 3 subtype, the conjugates OFH5503 and OFH611 could, due to their high affinity to the M 1 receptor, evolve as even more versatile tools in the field of research on muscarinergic receptors.

Strategies enabling the pH-dependent conformational switching of amide bonds from trans to cis , and vice versa, are yet limited in the sense that, in a suitable pH range, one rotamer may be stabilized to a large extent while the complementary pH range only leads to a mixture of isomers. By exploiting the effects of steric demand and the interaction of the amide carbonyl with a positive charge, we herein present the first examples for reversible pH-dependent switching from full trans to full cis .

From the variety of methods known for the depolymerization of organosolv lignin, a broad range of diversely substituted aromatic compounds are available today. In the present work, a novel two-step reaction sequence is reported, which is focused on the formation of phenols. While the first step of the depolymerization strategy comprises the 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ)-catalyzed oxidation of organosolv lignin with nitrogen monoxide so that two waste materials are combined, cleavage to the phenolic target compounds is achieved in the second step employing hydrazine and potassium hydroxide under Wolff-Kishner-type conditions. Besides the fact that the novel strategy proceeds via an untypical form of oxidized organosolv lignin, the two-step sequence is further able to provide phenols as cleavage products, which bear no substituent at the 4-position., Competing Interests: The authors declare no competing financial interest., (© 2021 The Authors. Published by American Chemical Society.)

The synthesis of pyridazinium salts was achieved from readily available phenylazosulfonates in a single reaction step. The reaction proceeds via the formation of short-lived phenyldiazenes, which-owing to the strongly acidic conditions-are partially protonated. The phenyldiazenes then undergo a rapid cycloaddition to furans to give pyridazinium salts via elimination of water. The fact that the pyridazinium synthesis shows a low sensitivity toward oxygen, although phenyldiazenes occur as intermediates, can be explained by the very fast cycloaddition step and the partial protonation of the phenyldiazene.

As a novel Sanger-type reagent, 2-fluoro-5-nitrophenyldiazonium tetrafluoroborate enabled the versatile functionalization of primary and secondary aliphatic alcohols. Based on a mild nucleophilic aromatic substitution of the fluorine atom under unprecedented, base-free conditions, the diazonium unit on the aromatic core of the resulting aryl-alkyl ether could be employed for such diverse transformations as radical C-H activation and cyclization, as well as palladium catalyzed cross-coupling reactions., (© 2021 The Authors. Published by Wiley-VCH GmbH.)

Metal and catalyst-free carbohydroxylations and carboetherifications at room temperature have been achieved by a combination of beneficial factors including high aryl diazonium concentration and visible light irradiation. The acceleration of the reaction by visible light irradiation is particularly remarkable against the background that neither the aryldiazonium salt nor the alkene show absorptions in the respective range of wavelength. These observations point to weak charge transfer interactions between diazonium salt and alkene, which are nevertheless able to considerably influence the reaction course. As highly promising perspective, many more aryldiazonium-based radical arylations might benefit from simple light irradiation without requiring a photocatalyst or particular additive., (© 2020 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH.)

Taking advantage of the "differentiating effect" of the solvent methanol, deuterations of electron-rich aromatic systems can be carried out under mild acid catalysis and thus under far milder conditions than known so far. The exceptional functional group tolerance observed under the optimized conditions, which even includes highly acid-labile groups, results from a hitherto unexploited shifted selectivity in protonation, and enabled simple and straightforward access to complex deuterium-labeled compounds.

The oxidation of alkyl thiols to disulfides has been achieved under mild conditions using a chemiluminescent 1,2-dioxetane as a stoichiometric oxidant. Besides the mild and biocompatible reaction conditions, this approach offers the possibility to monitor the presence of thiols through oxidation and chemiluminescence of the remaining dioxetane.

Targeted structural modifications have led to a novel type of buprenorphine-derived opioid receptor ligand displaying an improved selectivity profile for the μ-OR subtype. On this basis, it is shown that phenylazocarboxamides may serve as useful bioisosteric replacements for the widely occurring cinnamide units, without loss of OR binding affinity or subtype selectivity. This study further includes functional experiments pointing to weak partial agonist properties of the novel μ-OR ligands, as well as docking and metabolism experiments. Finally, the unique bifunctional character of phenylazocarboxylates, herein serving as precursors for the azocarboxamide subunit, was exploited to demonstrate the accessibility of an 18 F-fluorinated analogue., (© 2020 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

Intracellular concentration of reactive oxygen species (e.g., H 2 O 2 ) in cancer cells is elevated over 10-fold as compared to normal cells. This feature has been used by us and several other research groups to design cancer specific prodrugs, for example, N -alkylaminoferrocene (NAAF)-based prodrugs. Further improvement of the efficacy of these prodrugs can be achieved by their targeting to intracellular organelles containing elevated reactive oxygen species (ROS) amounts. For example, we have previously demonstrated that lysosome-targeted NAAF-prodrugs exhibit higher anticancer activity in cell cultures, in primary cells and in vivo (Angew. Chem. Int. Ed. 2017, 56, 15545). Mitochondrion is an organelle, where electrons can leak from the respiratory chain. These electrons can combine with O 2 , generating O 2 -• that is followed by dismutation with the formation of H 2 O 2 . Thus, ROS can be generated in excess in mitochondria and targeting of ROS-sensitive prodrugs to these organelles could be a sensible possibility for enhancing their efficacy. We have previously reported on NAAF-prodrugs, which after their activation in cells, are accumulated in mitochondria (Angew. Chem. Int. Ed. 2018, 57, 11943). Now we prepared two hybrid NAAF-prodrugs directly accumulated in mitochondria and activated in these organelles. We studied their anticancer activity and mode of action. Based on these data, we concluded that ROS produced by mitochondria is not available in sufficient quantities for activation of the ROS-responsive prodrugs. The reason for this can be efficient scavenging of ROS by antioxidants. Our data are important for the understanding of the mechanism of action of ROS-activatable prodrugs and will facilitate their further development.

Muscarinic M 3 receptor antagonists and inverse agonists displaying high affinity and subtype selectivity over the antitarget M 2 are valuable pharmacological tools and may enable improved treatment of chronic obstructive pulmonary disease (COPD), asthma, or urinary incontinence. On the basis of known M 3 antagonists comprising a piperidine or quinuclidine unit attached to a biphenyl carbamate, 5-fluoro substitution was responsible for M 3 subtype selectivity over M 2 , while 3'-chloro substitution substantially increased affinity through a σ-hole interaction. Resultantly, two piperidinyl- and two quinuclidinium-substituted biphenyl carbamates OFH243 ( 13n ), OFH244 ( 13m ), OFH3911 ( 14n ), and OFH3912 ( 14m ) were discovered, which display two-digit picomolar affinities with K i values from 0.069 to 0.084 nM, as well as high selectivity over the M 2 subtype (46- to 68-fold). While weak inverse agonistic properties were determined for the biphenyl carbamates 13m and 13n , neutral antagonism was observed for 14m and 14n and tiotropium under identical assay conditions.

Aryl radicals generated in the aqueous phase of biphasic mixtures have-regardless of a comparably low polarity- a strong preference to react with aromatic substrates in the aqueous phase and not to undergo phase-transfer into a lipophilic phase, independent from the presence of a surfactant. These results represent an important prerequisite toward future studies in biological systems, which typically consist of various compartments of either hydrophilic or lipophilic character.

Many subtype-selective dopamine receptor ligands developed for the D 2 -D 4 family incorporate a 1-arylpiperazine-derived primary recognition motif, which is connected to a lipophilic moiety occupying an extended binding pocket (EBP) of the receptor via an aliphatic linker of variable lengths. The evaluation of a novel group of dopamine receptor ligands now showed that highly subtype-selective ligands [up to K i (D 4.4 ) = 0.25 nM, D 2L /D 4.4 = 320, D 3 /D 4.4 = 710 for APH199 ( 17 )] can be obtained by choosing a relatively large and conformationally flexible 1-benzyl-1-phenylsemicarbazide substructure to fill the EBP. The novel chemotype APH199 ( 17 ) was found to act as a full agonist at the D 4 receptor showing significant bias toward G protein activation over β-arrestin recruitment in comparison to quinpirole.

The development of versatile functionalization concepts for graphene is currently in the focus of research. Upon oxo-functionalization of graphite, the full surface of graphene becomes accessible for C-C bond formation to introduce out-of-plane functionality. Herein, we present the arylation of graphene with arylazocarboxylic tert-butyl esters, which generates aryl radicals after activation with an acid. Surprisingly, the degree of functionalization is related to the concentration of lattice vacancy defects in the graphene material. Consequently, graphene materials that are free from lattice defects are not reactive. The reaction can be applied to graphene dispersed in solvents and leads to bitopic functionalization as well as monotopic functionalization when the graphene is deposited on surfaces. As the arylazocarboxylic tert-butyl ester moiety can be attached to various molecules, the presented method paves the way to functional graphene derivatives, with the density of defects determining the degree of functionalization., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Radical carbofluorination reactions starting from arylhydrazines and nonactivated alkenes, in which the C-F bond is formed through the use of Selectfluor, can be improved through the addition of anisole. Because direct trapping products could be detected only in trace amounts, anisole does primarily act as a reversible scavenger for the highly reactive ammonium radical dication released from Selectfluor in the C-F bond-forming step. As shown for three diverse substitution patterns, the main role of anisole is to prevent, or at least reduce, the undesired addition of the ammonium radical dication to the alkene, which in turn leads to an unfavorable consumption of the arylhydrazine-derived precursors required for carbofluorination. Moreover, besides the remarkable polar effects in radical trapping, this study shows that the Selectfluor-derived nitrogen-centered radical dication may add directly to alkenes, which has not been described so far., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

In this work, a novel phenethylamine-derived protecting group is introduced, which is able to significantly enhance the Grubbs I-catalyzed formation of 9- to 12-membered lactams through charge-induced conformational fixation under acidic conditions. As the new approach is particularly valuable for 10- and 11-membered ring systems, for which no related precedence was available so far, the overall strategy now offers general access to medium-sized lactams via ring closing metathesis. Cleavage of the protecting group can be achieved through a mild sequence combining N-oxidation and Cope elimination or alternatively under standard hydrogenation conditions.

Drugs that treat chronic obstructive pulmonary disease by antagonizing the M3 muscarinic acetylcholine receptor (M3R) have had a significant effect on health, but can suffer from their lack of selectivity against the M2R subtype, which modulates heart rate. Beginning with the crystal structures of M2R and M3R, we exploited a single amino acid difference in their orthosteric binding pockets using molecular docking and structure-based design. The resulting M3R antagonists had up to 100-fold selectivity over M2R in affinity and over 1,000-fold selectivity in vivo. The crystal structure of the M3R-selective antagonist in complex with M3R corresponded closely to the docking-predicted geometry, providing a template for further optimization., Competing Interests: Conflict of interest statement: B.K.K. is a cofounder of and consultant for ConfometRx, Inc., (Copyright © 2018 the Author(s). Published by PNAS.)

Our recent explorations of allosteric modulators with improved properties resulted in the identification of two biased negative allosteric modulators, BD103 ( N -1-{[3-(4-ethoxyphenyl)-4-oxo-3,4-dihydropyrido[2,3- d ]pyrimi-din2yl]ethyl}-4-(4-fluorobutoxy)- N -[(1-methylpiperidin-4-yl)methyl}]butanamide) and BD064 (5-[( N -{1-[3-(4-ethoxyphenyl)-4-oxo-3,4-dihydropyrido[2,3- d ]pyrimidin-2-yl]ethyl-2-[4-fluoro-3-(trifluoromethyl)phenyl]acetamido)methyl]-2-fluorophenyl}boronic acid), that exhibited probe-dependent inhibition of CXC-motif chemokine receptor CXCR3 signaling. With the intention to elucidate the structural mechanisms underlying their selectivity and probe dependence, we used site-directed mutagenesis combined with homology modeling and docking to identify amino acids of CXCR3 that contribute to modulator binding, signaling, and transmission of cooperativity. With the use of allosteric radioligand RAMX3 ([ 3 H] N -{1-[3-(4-ethoxyphenyl)-4-oxo-3,4-dihydropyrido[2,3- d ]pyrimidin-2-yl]ethyl}-2-[4-fluoro-3-(trifluoromethyl)phenyl]- N -[(1-methylpiperidin-4-yl)methyl]acetamide), we identified that F131 3.32 and Y308 7.43 contribute specifically to the binding pocket of BD064, whereas D186 4.60 solely participates in the stabilization of binding conformation of BD103. The influence of mutations on the ability of negative allosteric modulators to inhibit chemokine-mediated activation (CXCL11 and CXCL10) was assessed with the bioluminescence resonance energy transfer-based cAMP and β -arrestin recruitment assay. Obtained data revealed complex molecular mechanisms governing biased and probe-dependent signaling at CXCR3. In particular, F131 3.32 , S304 7.39 , and Y308 7.43 emerged as key residues for the compounds to modulate the chemokine response. Notably, D186 4.60 , W268 6.48 , and S304 7.39 turned out to play a role in signal pathway selectivity of CXCL10, as mutations of these residues led to a G protein-active but β -arrestin-inactive conformation. These diverse effects of mutations suggest the existence of ligand- and pathway-specific receptor conformations and give new insights in the sophisticated signaling machinery between allosteric ligands, chemokines, and their receptors, which can provide a powerful platform for the development of new allosteric drugs with improved pharmacological properties., (Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.)

A new type of waste recycling strategy is described in which nitrogen oxides or nitric acid are directly employed in photocatalyzed hydroxylations and nitrohydroxylations of benzenes. Through these transformations, otherwise costly denitrification can be combined with the synthesis of valuable compounds for various applications.

18 F-Labeled building blocks from the type of [ 18 F]fluorophenylazocarboxylic- tert -butyl esters offer a rapid, mild, and reliable method for the 18 F-fluoroarylation of biomolecules. Two series of azocarboxamides were synthesized as potential radioligands for dopamine D3 and the μ-opioid receptor, revealing compounds 3d and 3e with single-digit and sub-nanomolar affinity for the D3 receptor and compound 4c with only micromolar affinity for the μ-opioid receptor, but enhanced selectivity for the μ-subtype in comparison to the lead compound AH-7921. A "minimalist procedure" without the use of a cryptand and base for the preparation of 4-[ 18 F]fluorophenylazocarboxylic- tert -butyl ester [ 18 F]2a was established, together with the radiosynthesis of methyl-, methoxy-, and phenyl-substituted derivatives ( [ 18 F]2b-f ). With the substituted [ 18 F]fluorophenylazocarbylates in hand, two prototype azocarboxylates radioligands were synthesized by 18 F-fluoroarylation, namely the methoxy azocarboxamide [ 18 F]3d as the D3 receptor radioligand and [ 18 F]4a as a prototype structure of the μ-opioid receptor radioligand. By introducing the new series of [ 18 F]fluorophenylazocarboxylic- tert -butyl esters, the method of 18 F-fluoroarylation was significantly expanded, thereby demonstrating the versatility of 18 F-labeled phenylazocarboxylates for the design of potential radiotracers for positron emission tomography ., Competing Interests: The authors declare no competing financial interest.

Substituted indoles can be prepared from phenylazocarboxylates through a rapid one-pot sequence featuring a microwave-assisted Fischer indole synthesis as a key step. Considering that the phenylazocarboxylates may beforehand be modified by mild nucleophilic aromatic substitution, including the introduction of [ 18 F]fluoride, the overall strategy offers an attractive new access to 5-[ 18 F]fluoroindoles., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

In the absence of a photocatalyst and other additives, the radical arylation of diverse arenes and heteroarenes has been achieved with aryldiazonium salts under visible-light irradiation from a blue light-emitting diode (LED). Although the course of some reactions can be rationalized by the formation of strongly light-absorbing charge-transfer (CT) complexes between the diazonium ion and the aromatic substrate, several further examples indicated that the simple presence of an aromatic substrate, showing only weak interactions to the diazonium ion, is fully sufficient to enable product formation., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The radical arylation of anilines and pyrroles can be achieved under transition-metal- and catalyst-free conditions by using aryldiazotates in strongly alkaline aqueous solutions. The aryldiazotates act as protected diazonium ions, which do not undergo azo coupling with electron-rich aromatic substrates, but can still serve as an aryl radical source at slightly elevated temperatures. Based on an improved preparation of aryldiazotates in aqueous solution, homolytic aromatic substitutions of anilines and pyrroles were conducted with good overall yields and high regioselectivity. Moreover, DFT calculations provided further mechanistic insights., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The radical 5-exo cyclization starting from 2-allyloxyphenyldiazonium ions can be employed for the diastereoselective synthesis of ortho-spirocyclohexadienones through a consecutive addition to alkynes. The spirocyclic systems are formed in a radical [2 + 2 + 1] cycloaddition comprising three carbon-carbon formations, of which the final one is an ipso attack onto the aromatic system at the original position of the diazonium-derived aryl radical.

Low concentrations of nitrogen dioxide, which arises as a side product from a range of industrial processes, can effectively be recycled through the diazotization of anilines. The studies reported herein now demonstrate that the removal of nitrogen dioxide from gas streams is even more effective when hydrophilic anilines are used as starting materials. The diazonium salts, which are obtained in this way in up to quantitative yields, can directly be employed in azo coupling reactions, thus opening up an attractive route to the industrially important group of azo compounds., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Although general interest in radical arylation reactions has grown rapidly in recent years, poor regioselectivities and the need to use a large excess of the radical-accepting arene have hindered their application to substituted benzenes. We now describe experimental and computational investigations into the substituent effects that lead to regioselective addition based on the recent discovery of anilines as outstanding substrates for radical arylations.

Overexpression of the histone lysine demethylase KDM4A, which regulates H3K9 and H3K36 methylation states, has been related to the pathology of several human cancers. We found that a previously reported hydroxamate-based histone deacetylase (HDAC) inhibitor (SW55) was also able to weakly inhibit this demethylase with an IC50 value of 25.4 μm. Herein we report the synthesis and biochemical evaluations, with two orthogonal in vitro assays, of a series of derivatives of this lead structure. With extensive chemical modifications on the lead structure, also by exploiting the versatility of the radical arylation with aryldiazonium salts, we were able to increase the potency of the derivatives against KDM4A to the low-micromolar range and, more importantly, to obtain demethylase selectivity with respect to HDACs. Cell-permeable derivatives clearly showed a demethylase-inhibition-dependent antiproliferative effect against HL-60 human promyelocytic leukemia cells., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The radical carbohydroxylation of styrenes with aryldiazonium salts has been achieved under mild thermal conditions. A broad range of aryldiazonium salts was tolerated, and the reaction principle based on a radical-polar crossover mechanism could be extended to carboetherification as well as to a two-step, metal-free variant of the Meerwein arylation leading to stilbenes., (© 2016 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

The titanium(III)-mediated radical arylation of 3-hydroxypyridines was found to proceed with high regioselectivity for the 2-position. Using aryldiazonium chlorides, which were prepared from the corresponding anilines, as aryl radical sources, a range of 3-hydroxy-2-phenylpyridines were obtained in moderate to good yields under simple reaction conditions. Reactions of ortho-carboxylic ester substituted phenyldiazonium salts directly provided tricyclic benzopyranopyridinones.

Mizoroki-Heck reactions, which are well-known for aryldiazonium salts and which have recently been described for arylhydrazines, have now been extended to phenyldiazenes. In situ generation of phenyldiazenes from azocarboxylates allowed clean and selective reactions with styrenes, acrylates, and acrylamides using palladium(II) acetate in the presence of silver(I) acetate or hydrogen peroxide as oxidant. Hydrogen peroxide was thereby shown to be a cheap and broadly applicable alternative for the established palladium-silver(I) system.

The CXCR3 receptor, a class A G protein-coupled receptor (GPCR), is involved in the regulation and trafficking of various immune cells. CXCR3 antagonists have been proposed to be beneficial for the treatment of a wide range of disorders including but not limited to inflammatory and autoimmune diseases. The structure-based design of CXCR3 ligands remains, however, hampered by a lack of structural information describing in detail the interactions between an allosteric ligand and the receptor. We designed and synthesized photoactivatable probes for the structural and functional characterization, using photoaffinity labeling followed by mass spectrometry, of the CXCR3 allosteric binding pocket of AMG 487 and RAMX3, two potent and selective CXCR3 negative allosteric modulators. Photoaffinity labeling is a common approach to elucidate binding modes of small-molecule ligands of GPCRs through the aid of photoactivatable probes that convert to extremely reactive intermediates upon photolysis. The photolabile probe N-[({1-[3-(4-ethoxyphenyl)-4-oxo-3,4-dihydropyrido[2,3-d]pyrimidin-2-yl]ethyl}-2-[4-fluoro-3-(trifluoromethyl)phenyl]-N-{1-[4-(3-(trifluoromethyl)-3H-diazirin-3-yl]benzyl}piperidin-4-yl)methyl]acetamide (10) showed significant labeling of the CXCR3 receptor (80%) in a [(3) H]RAMX3 radioligand displacement assay. Compound 10 will serve as an important tool compound for the detailed investigation of the binding pocket of CXCR3 by mass spectrometry., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Distinct diaminopyrimidines, for example, 4-(4-methylpiperazin-1-yl)-5,6-dihydrobenzo[h]quinazolin-2-amine are histamine H4 receptor (H4R) antagonists and show high affinity to the H4R, but only a moderate affinity to the histamine H1 receptor (H1R). Within previous studies it was shown that an aromatic side chain with a distinct distance to the basic amine and aromatic core is necessary for affinity to the human H1R (hH1R). Thus, a rigid aminopyrimidine with a tricyclic core was used as a lead structure. There, (1) the flexible aromatic side chain was introduced, (2) the substitution pattern of the pyrimidine core was exchanged and (3) rigidity was decreased by opening the tricyclic core. Within the present study, two compounds with similar affinity in the one digit μM range to the human H1R and H4R were identified. While the affinity at the hH1R increased about 4- to 8-fold compared to the parent diaminopyrimidine, the affinity to the hH4R decreased about 5- to 8-fold. In addition to the parent diaminopyrimidine, two selected compounds were docked into the H1R and H4R and molecular dynamic studies were performed to predict the binding mode and explain the experimental results on a molecular level. The two new compounds may be good lead structures for the development of dual H1/H4 receptor ligands with affinities in the same range., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

A new transition-metal-free version of the Meerwein arylation has been developed. The key feature of this carboamination-type reaction is the slow base-controlled generation of aryl radicals from aryldiazonium tetrafluoroborates, so that a sufficient quantity of diazonium ions remains to enable efficient trapping of the alkyl radical adduct resulting from aryl radical addition to the alkene. Under strongly basic conditions, diazoanhydrides are likely to take over the role of the nitrogen-centered radical scavengers.

Allylation and benzylation of phenylazocarboxylic tert-butyl esters have been achieved under Barbier-type reaction conditions and in very short reactions times using the corresponding allyl and benzyl bromides or iodides in combination with zinc powder. Whereas all reactions occurred exclusively at the β-nitrogen atom of the azocarboxylic esters, the linkage of allyl units was shown to depend on the substitution pattern at the double bond of the allyl halide. The hydrazines obtained are useful precursors for indoles and indazoles.

The oxidative nitration of styrenes in ethyl acetate represents a metal-free, environmentally friendly, and sustainable technique to recover even low concentrations of NO2 in air. Favorable features are that the product mixture comprising nitroalcohols, nitroketones, and nitro nitrates simplifies at lower concentrations of NO2 . Experiments in a miniplant-type 10 L wet scrubber demonstrated that the recycling technique is well applicable on larger scales at which initial NO2 concentrations of >10 000 ppm were reliably reduced to less than 40 ppm., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Tertiary amides, which usually occur as cis/trans mixtures, can be effectively shifted to the cis conformation by placing a positive charge in close proximity to the amide carbonyl. This effect was used to prepare cis-configured prolyl amides and to facilitate a strongly rotamer-dependent radical cyclization., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Phenylazocarboxamides can serve as bioisosteres for cinnamides, which are widely occurring substructures in medicinal chemistry. Starting from our lead compound 2, the introduction of additional fluoro substituents and the exchange of the methoxyphenylpiperazine head group by an aminoindane moiety was investigated resulting in dopamine D3 receptor antagonists and agonists with Ki values in the sub- and low-nanomolar range. As a potentially irreversible ligand, the 3,4,5-trifluoro-substituted phenylazocarboxamide 7 was investigated for its N-arylating properties by incubation with the protected lysine analog 18 and with the L89K mutant of the dopamine D3 receptor. Whereas covalent bond formation with the lysine unit in TM2 of D3 could not be detected, substantial N-arylation of the side chain of the model compound 18 has been observed., (Copyright © 2014 Elsevier Ltd. All rights reserved.)