On the Mechanism of the Formal [2+2] Cycloaddition - Retro-electrocyclization (CA-RE) Reaction.

The [2+2] cycloaddition - retro-electrocyclization (CA-RE) reaction is a "click-like" protocol for facile synthesis of donor-acceptor chromophores from an alkyne and tetracyanoethylene. Herein we shed light on the mechanism of this reaction by detailed kinetics studies using 1 H NMR spectroscopy. By considering several experiments simultaneously, a variety of mechanistic models was evaluated. Surprisingly, a model in which the final 1,1,4,4-tetracyanobuta-1,3-diene product promoted the first step was the only one that described well the experimental data. This autocatalysis model also involved a non-concerted, stepwise formation of the cyclobutene cycloaddition adduct. By proper choice of conditions, we were able to generate the transient cyclobutene in sufficient amount to verify it as an intermediate using 13 C NMR spectroscopy. For its final retro-electrocyclization step, simple first-order kinetics was observed and only minor solvent dependence, which indicates a concerted reaction.

Identification of the synthetic cannabinoid-type new psychoactive substance, CH-PIACA, in seized material.

Synthetic cannabinoids (SCs) remain the largest class of new psychoactive substances (NPS), and while the number of NPS that are reported to the European Monitoring Centre for Drugs and Drug Addiction (EMCDDA) for the first time each year declines, the number of newly reported SCs still exceeds other NPS classes. This decline can be seen as a result of legislative changes by different jurisdictions which have sometimes transitioned to a more generalized approach when controlling substances by defining common structural scaffolds rather than explicit structures. While the consequences of such legislative changes have been expected over the years, the introduction of so-called "class-wide" bans puts further pressure on clandestine laboratories to synthesize compounds which are out of the scope of the legislation, and thus, these compounds are initially harder to detect and/or identify in the absence of analytical data. Recently, a SC with an indole-3-acetamide core-linker scaffold, AD-18 (i.e., ADB-FUBIATA or ADB-FUBIACA), was reported for the first time in China in 2021. Here, an additional cannabinoid with the indole-3-acetamide scaffold, N-cyclohexyl-2-(1-pentyl-1H-indol-3-yl)acetamide (CH-PIACA), is reported which was identified for the first time in a seized material in Denmark. Structural characterization was performed using gas chromatography-mass spectrometry (GC-MS), liquid chromatography-high-resolution mass spectrometry (LC-HRMS), and nuclear magnetic resonance (NMR) spectroscopy.

Orthogonal Photoswitching with Norbornadiene.

Orthogonal photoswitching is a convenient but challenging way of controlling multiple functions in a system by selective photoisomerization of one unit before the other in any arbitrarily chosen sequence. Here, we present this concept for the norbornadiene/quadricyclane (NBD/QC) photo/thermo-switch in the presence or absence of a coordinated metal ion. Thus, introducing two pyridyl ligands via ethyne-1,2-diyl bridges provides a system that by chelation of metal ions, such as PdII , has altered optical and switching properties. Mixing the PdII complex with its free ligand furnishes a four-state system where NBD-to-QC photoisomerizations for complexed and uncomplexed species are controlled by the irradiation wavelength and can occur orthogonally, that is, the sequence of photoisomerizations can be swapped. Studies on AgI and PbII complexes, being less stable than the PdII complex, are also presented; these exhibit like the PdII complex significantly red-shifted NBD absorptions.

Complexation of Fullerenes by Subphthalocyanine Dimers.

Tweezer-like molecules comprised of two boron subphthalocyanine (SubPc) units were prepared by Sonogashira couplings and investigated using NMR spectroscopy for their ability to bind fullerenes (C60 and C70). The preorganization of the tweezers provided association constants of ca. 103 M-1 in toluene- d8, while a SubPc monomer did not show any association. Nevertheless, the SubPc monomer crystallized with the fullerenes as 2:1 complexes, supporting the favorable tweezer-like design for complexation, which was further corroborated by computations.

Azulenium chemistry: towards new derivatives of photochromic dihydroazulenes.

Here we present the preparation of a selection of azulenium ions by hydride abstraction from photochromic 1,8a-dihydroazulenes (1,8a-DHAs) incorporating two cyano groups at C-1. The reactivity of the electrophilic tropylium ring of these molecules towards lithium triisopropylsilylacetylide was investigated. The position of attack by the nucleophile depended on the substitution pattern of the azulenium cation but was in general found to occur preferentially at positions C-4, C-5, and C-6, and to a minor extent at positions C-7 and C-8. The outcome was a mixture of non-photochromic, regioisomeric DHAs. One of these compounds containing the ethynyl substituent at position C-4 was partly tautomerized to the photochromic 1,8a-dihydroazulene, which was isolated and its switching properties were investigated by UV-Vis absorption spectroscopy. Upon irradiation, it undergoes a ring-opening reaction to form a vinylheptafulvene (VHF), which in turn returns to the original DHA. The half-life of this reaction was significantly smaller than for a derivative with the alkynyl substituent placed at C-7. This fast switching behavior was according to the calculations explained by an enhancement in the stability of the reactive s-cis conformer of the VHF relative to the, still more stable, s-trans conformer, and by a smaller activation energy for this s-cis conformer to undergo ring-closure.

Synthesis and properties of unsymmetrical azatrioxa[8]circulenes.

Insights to the subtle reactivity patterns of hydroxy-substituted carbazoles allows the precise synthesis of unsymmetrical azatrioxa[8]circulenes by the reaction of N-benzyl-2,7-di-tert-butyl-3,6-dihydroxycarbazole with two different 1,4-benzoquinones in the presence of an oxidant (chloranil) and a Lewis acid (BF3OEt2). The unique synthetic control obtained originates from the selectivity obtained upon reacting N-benzyl-2,7-di-tert-butyl-3,6-dihydroxycarbazole with an electron-rich benzoquinone to give first the C-C bond formation and then subsequently the dibenzofuran formation with high regioselectivity. Herein the first synthesis of unsymmetrical antiaromatic azatrioxa[8]circulenes and the full characterization using NMR spectroscopy, optical spectroscopy, electrochemistry, computational techniques and single crystal X-ray crystallography is reported. The controlled stepwise condensation of N-benzyl-2,7-di-tert-butyl-3,6-dihydroxycarbazole with two different 1,4-benzoquinones gives selectively the unsymmetrical azatrioxa[8]circulenes.

Cyanohydrin reactions enhance glycolytic oscillations in yeast.

Synchronous metabolic oscillations can be induced in yeast by addition of glucose and removal of extracellular acetaldehyde (ACAx). Compared to other means of ACAx removal, cyanide robustly induces oscillations, indicating additional cyanide reactions besides ACA to lactonitrile conversion. Here, (13)C NMR is used to confirm our previous hypothesis, that cyanide directly affects glycolytic fluxes through reaction with carbonyl-containing compounds. Intracellularly, at least 3 cyanohydrins were identified. Extracellularly, all signals could be identified and lactonitrile was found to account for ~66% of total cyanide removal. Simulations of our updated computational model show that intracellular cyanide reactions increase the amplitude of oscillations and that cyanide addition lowers [ACA] instantaneously. We conclude that cyanide provides the following means of inducing global oscillations: a) by reducing [ACAx] relative to oscillation amplitude, b) by targeting multiple intracellular carbonyl compounds during fermentation, and c) by acting as a phase resetting stimulus.

2-(2-Bromophenyl)-formononetin and 2-heptyl-formononetin are PPARγ partial agonists and reduce lipid accumulation in 3T3-L1 adipocytes.

Isoflavones are bioactive compounds that have been shown to decrease lipid accumulation in vitro. However, the knowledge of the isoflavone formononetin is limited. The aim of the study was to assess the effects of formononetin and its two synthetic analogues, 2-(2-bromophenyl)-formononetin and 2-heptyl-formononetin, on lipid accumulation in 3T3-L1 adipocytes and investigate possible mechanisms. Formononetin and the two analogues were added day 0-8 or day 8-10 of the differentiation period, and lipid accumulation, glycerol release and gene expression were measured. Additionally, competitive peroxisome proliferator-activated receptor (PPAR)-γ binding assay, PPARγ transactivation assay and Western blot for phosphorylated AMP-activated protein kinase (AMPK) were performed. Chronic treatment (day 0-8) with formononetin increased lipid accumulation, whereas the two analogues decreased lipid accumulation partly due to decreased differentiation. The two analogues, but not formononetin, also decreased lipid content in mature adipocytes. 2-Heptyl-formononetin increased glycerol release and lipolytic gene expression and decreased lipogenic gene expression. Formononetin did not bind to or activate PPARγ whereas both analogues bound to the receptor and behaved as PPARγ partial agonists in the transactivation assay. Neither of the compounds affected phosphorylation of AMPK. In conclusion, the analogues of formononetin decreased lipid accumulation possibly in part by acting as PPARγ partial agonists.

2-heptyl-formononetin increases cholesterol and induces hepatic steatosis in mice.

Consumption of isoflavones may prevent adiposity, hepatic steatosis, and dyslipidaemia. However, studies in the area are few and primarily with genistein. This study investigated the effects of formononetin and its synthetic analogue, 2-heptyl-formononetin (C7F), on lipid and cholesterol metabolism in C57BL/6J mice. The mice were fed a cholesterol-enriched diet for five weeks to induce hypercholesterolemia and were then fed either the cholesterol-enriched diet or the cholesterol-enriched diet-supplemented formononetin or C7F for three weeks. Body weight and composition, glucose homeostasis, and plasma lipids were compared. In another experiment, mice were fed the above diets for five weeks, and hepatic triglyceride accumulation and gene expression and histology of adipose tissue and liver were examined. Supplementation with C7F increased plasma HDL-cholesterol thereby increasing the plasma level of total cholesterol. Supplementation with formononetin did not affect plasma cholesterol but increased plasma triglycerides levels. Supplementation with formononetin and C7F induced hepatic steatosis. However, formononetin decreased markers of inflammation and liver injury. The development of hepatic steatosis was associated with deregulated expression of hepatic genes involved in lipid and lipoprotein metabolism. In conclusion, supplementation with formononetin and C7F to a cholesterol-enriched diet adversely affected lipid and lipoprotein metabolism in C57BL/6J mice.

Synthesis and stability study of a new major metabolite of γ-hydroxybutyric acid.

γ-Hydroxybutanoic acid (GHB) is used as a date-rape drug, which renders the victims unconscious and defenceless. Intoxications are very difficult to detect for forensic scientists due to rapid metabolism to endogenous levels of GHB. We recently discovered a new major metabolite, 2, of GHB (1) that could potentially extend the analytical detection window for GHB intoxications. Herein we disclose synthetic procedures based on a Koenigs-Knorr glucuronidation approach that provides GHB glucuronide 2 and a deuterium-labelled analogue d 4-2 of high purity suitable for analytical chemistry. In addition, we have assessed the stability of GHB glucuronide 2 by mimicking the natural pH range for urine, which is of importance in the development of new analytical methods. Using NMR we show that GHB glucuronide 2 is highly stable towards aqueous hydrolysis within the pH range normally observed for urine even at elevated temperature.

Identification of a new metabolite of GHB: gamma-hydroxybutyric acid glucuronide.

Gamma-hydroxybutyric acid (GHB) is an important analyte in clinical and forensic toxicology with a narrow detection window of 3-6 h. In the search of improved detection methods, the existence in vivo of a glucuronated GHB metabolite (GHB-GLUC) was hypothesized. Chemically pure standards of GHB-GLUC and a deuterated analogue for chromatography were synthesized. Liquid chromatography and tandem mass spectrometry were used for targeted analysis in anonymous clinical urine samples (n = 50). GHB-GLUC was found in concentrations ranging from 0.11 to 5.0 µg/mL (mean: 1.3 ± 1.2 µg/mL). Thus far, this is the first report of a GHB glucuronide detected in biological samples. Given that glucuronides generally have longer half-life values than their corresponding free drugs, GHB-GLUC should theoretically be a biomarker of GHB intoxication. It is also proposed that the hitherto unexplained reports of elevated GHB concentrations in some biological samples, which has caused the setting of a relatively high cutoff value (10 µg/mL), represent total GHB measurements (sum of free GHB and actively chemically hydrolyzed GHB-GLUC). To address these challenges, the present study must be followed by comprehensive pharmacokinetic and stability studies after the controlled administration of GHB.

A bis(heptafulvenyl)-dicyanoethylene thermoswitch with two sites for ring closure.

Suitably functionalized vinylheptafulvenes (VHFs) act as thermoswitches undergoing ring closure to the corresponding dihydroazulenes (DHAs). Here we present the synthesis of a new such thermoswitch incorporating two heptafulvene rings on a dicyanoethylene unit. The synthetic protocol explores both the tropylium species as an electrophile and as a leaving group in the generation of the heptafulvene units. The thermally induced ring closure was enhanced as a result of two accessible sites for the reaction to occur.

Lewis acid enhanced switching of the 1,1-dicyanodihydroazulene/vinylheptafulvene photo/thermoswitch.

Mild Lewis acids enhance the rate of the thermal conversion of vinylheptafulvene (VHF) to dihydroazulene (DHA). In the absence of light, stronger Lewis acids promote the otherwise photoinduced DHA to VHF conversion.

Arylethynyl derivatives of the dihydroazulene/vinylheptafulvene photo/thermoswitch: tuning the switching event.

A selection of dihydroazulene (DHA) photoswitches incorporating an arylethynyl-substituent in the seven-membered ring was prepared by palladium-catalyzed Sonogashira cross-coupling reactions employing a suitable bromo-functionalized DHA. Shielding of the alkyne bridge and separating the aryl and DHA units, by sterically demanding groups, was required to obtain stable compounds. The DHAs underwent a light-induced ring-opening to vinylheptafulvenes (VHFs) which were thermally converted to a mixture of two DHA regioisomers, one of which was the original one. The influence of the aryl groups on the DHA and VHF absorptions and on their interconversion was investigated in detail. The rates of the switching events were finely tuned by the donor or acceptor strength of the aryl group. The thermal ring closure was found to proceed most readily in the presence of an electron-donating group on the seven-membered ring. The rate constant was found to follow a Hammett linear free energy correlation, which signals that stabilization of a positive charge in the seven-membered ring plays a crucial role in the ring-closure reaction. In view of these findings, it was possible to control the switching event by protonation/deprotonation of an anilino-substituted DHA. Also, the light-induced ring opening reaction was strongly controlled by acid/base. In addition to the mesomeric effects exerted by an arylethynyl group, the inductive effects exerted by different groups on the thermal ring closure were elucidated. Although the alkyne bridge transmits the electronic character of the aryl group, the ring-closure is retarded for all the ethynylated compounds relative to the parent unsubstituted compound. Along with our synthesis of suitable arylalkynes, we discovered an interesting byproduct in a Sonogashira cross-coupling reaction involving a nitrophenyl group, namely a diaryl azoxy compound. Its structure was confirmed by X-ray crystallographic analysis.