Compound 1 is a selective and potent agonist of the G protein-coupled receptor GPR139 (EC 50 = 39 nM). In this study, we describe the synthesis, radiolabelling and in vitro evaluation of 3 H- 1 for ...the characterisation of GPR139 and its spatial expression in the brain using autoradiography. Two different synthesis routes for the radiolabelling of 1 based on a reductive debromination strategy were investigated using deuterium (D 2 , g). The route based on reductive debromination of the bromonaphthyl precursor 5 proved superior over arylbromide 4 and was employed for the radiolabelling experiments. Reductive debromination of precursor 5 was accomplished using 3 H 2 , Pd/C and triethylamine in DMF at ambient temperature to give target molecule 3 H- 1 with a specific activity of 19.3 Ci mmol −1 and a radiochemical purity of ≥95%. By application of autoradiography and binding studies, it was not possible to discriminate 3 H- 1 binding to wildtype mice brains from GPR139 knockout mice brains and total binding from non-specific binding in CHO-k1 cells stably expressing human GPR139 receptor. Based on these experiments we conclude that 3 H- 1 is not a suitable radioligand for the characterisation of GPR139.
3‐Hydroxycyclopent‐1‐ene‐1‐carboxylic acid (HOCPCA (1)) is a potent ligand for high‐affinity γ‐hydroxybutyric acid binding sites in the central nervous system. Various approaches to the introduction ...of a hydrogen label onto the HOCPCA skeleton are reported. The outcomes of the feasible C─H activation of olefin carbon (C‐2) by iridium catalyst are compared with the reduction of the carbonyl group (C‐3) by freshly prepared borodeuterides. The most efficient iridium catalysts proved to be Kerr bulky phosphine N‐heterocyclic species providing outstanding deuterium enrichment (up to 91%) in a short period of time. The highest deuterium enrichment (>99%) was achieved through the reduction of ketone precursor 2 by lithium trimethoxyborodeuteride. Hence, analogical conditions were used for the tritiation experiment. 3H‐HOCPCA selectively labeled on the position C‐3 was synthetized with radiochemical purity >99%, an isolated yield of 637 mCi and specific activity = 28.9 Ci/mmol.
Various approaches to the introduction of a hydrogen label onto the HOCPCA skeleton are reported. The outcomes of the feasible C─H activation of olefin carbon (C‐2) by iridium catalyst are compared with the reduction of the carbonyl group (C‐3) by freshly prepared borodeuterides.
Insulin-like growth factors 2 and 1 (IGF2 and IGF1) and insulin are closely related hormones that are responsible for the regulation of metabolic homeostasis, development and growth of the organism. ...Physiological functions of insulin and IGF1 are relatively well-studied, but information about the role of IGF2 in the body is still sparse. Recent discoveries called attention to emerging functions of IGF2 in the brain, where it could be involved in processes of learning and memory consolidation. It was also proposed that these functions could be mediated by the receptor for IGF2 (IGF2R). Nevertheless, little is known about the mechanism of signal transduction through this receptor. Here we produced His-tagged domain 11 (D11), an IGF2-binding element of IGF2R; we immobilized it on the solid support through a well-defined sandwich, consisting of neutravidin, biotin and synthetic anti-His-tag antibodies. Next, we prepared specifically radiolabeled .sup.125 I-monoiodotyrosyl-Tyr2-IGF2 and optimized a sensitive and robust competitive radioligand binding assay for determination of the nanomolar binding affinities of hormones for D11 of IGF2. The assay will be helpful for the characterization of new IGF2 mutants to study the functions of IGF2R and the development of new compounds for the treatment of neurological disorders.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The receptor channel transient receptor potential vanilloid 1 (TRPV1) functions as a sensor of noxious heat and various chemicals. There is increasing evidence for a crosstalk between TRPV1 and ...opioid receptors. Here we investigated the effect of the prototypical TRPV1 agonist capsaicin and selected opioid ligands on TRPV1 movement in the plasma membrane and intracellular calcium levels in HEK293 cells expressing TRPV1 tagged with cyan fluorescent protein (CFP). We observed that lateral mobility of TRPV1 increased after treatment of cells with capsaicin or naloxone (a nonselective opioid receptor antagonist) but not with DAMGO (a μ-opioid receptor agonist). Interestingly, both capsaicin and naloxone, unlike DAMGO, elicited intracellular calcium responses. The increased TRPV1 movement and calcium influx induced by capsaicin and naloxone were blocked by the TRPV1 antagonist capsazepine. The ability of naloxone to directly interact with TRPV1 was further corroborated by 3H-naloxone binding. In conclusion, our data suggest that besides acting as an opioid receptor antagonist, naloxone may function as a potential TRPV1 agonist.
Fast and reasonable low‐scale (200 nmol) syringe‐made synthesis of
15
N‐labeled oligonucleotides representing DNA trinucleotide codons is communicated. All codons were prepared by solid‐phase ...controlled pore glass synthesis column technique via the phosphoramidite method. Twenty‐four labeled oligonucleotides covering the DNA genetic code alphabet were prepared using commercially available reagents and affordable equipment in a reasonably short period of time, with acceptable yields and purity for direct applications in mass spectrometry.
Abstract
Insulin-like Growth Factor-2 (IGF2) is important for the regulation of human embryonic growth and development, and for adults’ physiology. Incorrect processing of the IGF2 precursor, ...pro-IGF2(156), leads to the formation of two IGF2 proforms, big-IGF2(87) and big-IGF2(104). Unprocessed and mainly non-glycosylated IGF2 proforms are found at abnormally high levels in certain diseases, but their mode of action is still unclear. Here, we found that pro-IGF2(156) has the lowest ability to form its inactivating complexes with IGF-Binding Proteins and has higher proliferative properties in cells than IGF2 and other IGF prohormones. We also showed that big-IGF2(104) has a seven-fold higher binding affinity for the IGF2 receptor than IGF2, and that pro-IGF2(87) binds and activates specific receptors and stimulates cell growth similarly to the mature IGF2. The properties of these pro-IGF2 forms, especially of pro-IGF2(156) and big-IGF2(104), indicate them as hormones that may be associated with human diseases related to the accumulation of IGF-2 proforms in the circulation.
Direct Multi‐Deuterium Labelling of Pirtobrutinib Kriegelstein, Michal; Hojcsková, Jana; Hroch, Miloš ...
Journal of labelled compounds & radiopharmaceuticals,
July 2024, 2024-07-00, 20240701, Letnik:
67, Številka:
9
Journal Article
Recenzirano
ABSTRACT
Herein, we demonstrate an efficient method for multi‐deuterium labelling of pirtobrutinib—a Bruton's tyrosine kinase inhibitor recently approved by the FDA—using a straightforward hydrogen ...isotope exchange (HIE) reaction. A remarkably high level of deuterium incorporation was achieved using an excess of a Kerr‐type iridium catalyst. The key factor in the significant deuterium labelling was the decision to employ a deuterium uniformly labelled solvent, chlorobenzene‐d5, at an elevated temperature. Virtually, no d0–d3 species were detected, with only traces of d4–d5 isotopomers (< 5%) observable in the mass spectrum of pirtobrutinib‐d8, fulfilling requirements for stable isotope‐labelled internal standard. The labelled compound—mainly consisting of isotopomers d6–d9 at 82.4% of the total abundance—was isolated in a high yield (73%) and purity (99%). Noteworthy, fluorine group acting as a directing group was observed for the first time. Significant incorporation of deuterium in ortho‐positions, exceeding 87%, was observed. Interestingly, chlorinated solvent used in the HIE reactions was non‐specifically deuterated yielding up to 0.42 deuterium per chlorobenzene molecule even at an exceptionally low iridium catalyst loading of 4.17 × 10–2 mol%.
We present an efficient method for multi‐deuterium labelling of pirtobrutinib—a BTK inhibitor recently approved by the FDA—using a straightforward hydrogen isotope exchange (HIE) reaction. The use of a deuterium‐labelled solvent of chlorobenzene‐d5 at elevated temperatures resulted in a high degree of deuterium labelling, mainly pirtobrutinib‐d8. Our results show that the fluorine moiety can act as a potent ortho‐directing group, selectively moderating HIE reactions in the aromatic system under specific conditions. We also observed significant deuterium labelling of the chlorobenzene solvent during the HIE reactions.
Reactivity of 2‐(4‐hydroxyphenyl)‐1H‐imidazoline and 2‐(4‐hydroxyphenyl)‐1H‐imidazole toward substituted phenyl isocyanates was studied. When mentioned imidazoline was treated with 2.5 equiv of ...substituted phenyl isocyanate, three N,O‐dicarboxamides were prepared (substituents are H, 4‐NO2, and 4‐CH3). Subsequently, N,O‐diacetylated 2‐(4‐hydroxyphenyl)‐1H‐imidazoline was prepared and selective deprotection method was developed for preparation of 1‐acetyl‐2‐(4‐hydroxyphenyl)‐1H‐imidazoline using diethylamine in acetone. Six carbamates derived from this imidazoline were then prepared using 1.1 equiv of substituted phenyl isocyanates (substituents are H, 4‐CH3, 4‐OCH3, 4‐NO2, 4‐CN, and 3‐CF3). Finally, two carbamates were prepared from 2‐(4‐hydroxyphenyl)‐1H‐imidazole (substituents are 4‐NO2 and 4‐CN). No reactivity to imidazole ring was observed in this case. Eight derivatives were subjected to antimycobacterial screening. Concurrently, reactivity of 2‐(2‐aminophenyl)‐ and 2‐(2‐hydroxyphenyl)‐1H‐imidazole toward aliphatic and aromatic isocyanates was studied. Eight ureas were prepared using equivalent mixture of 2‐(2‐aminophenyl)‐1H‐imidazole and isocyanate (Et, Pr, isoPr, terc‐Bu, Cy, Ph, 4‐CH3C6H4, 4‐CNC6H4). Similar attempts to obtain related carbamates from 2‐(2‐hydroxyphenyl)‐1H‐imidazole lead only to three substituted phenyl carbamates (substituents are 4‐CH3, 4‐NO2, and 4‐CN). In both cases, no reactivity to imidazole ring was observed again.
Reactive magnetron sputtering is a well-established technique to deposit compound films with different composition. Target poisoning influences the process stability and gives rise to hysteresis ...effects. Recently, a double S-shaped hysteresis curve has been reported, meaning two values of discharge voltage correspond to one defined value of the reactive gas partial pressure. In the present study, the long-term stability of the hysteresis behavior was studied in an industrial system equipped with a mass spectrometer, an optical emission spectrometer and an external anode. Cycles of decreasing and increasing the target voltage have been repeated several times for single Al alloy target DC sputtering and bipolar pulsed sputtering. In the first case, changes in the hysteresis S-shaped curve depending on time were detected indicating that the system moves towards the poisoned mode meaning higher currents at low voltages and vice versa. When an external additional anode is opened, the system instantly reacts by moving closer to the metallic mode. All of our observations indicate that the long-term behavior is caused by the effect of a disappearing anode and corresponding effects on the plasma medium. Bipolar sputtering reduces considerably the effects of disappearing anode, but a long time evolution can still be seen.
•Long-term behavior in DC is influenced by the effect of disappearing anode.•Disappearing anode results in a higher voltage for same oxygen partial pressure.•In DC, the target current changes when an additional clean anode is provided.•The disappearing anode effect was not detected in pulsed bipolar sputtering.
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