New developments in the synthesis of pyrrolizidinone-based dipeptide isosteres

1,3-Dipolar cycloaddition of acrylamide with the cyclic nitrone derived from proline tert-butyl ester has been employed in the synthesis of bicyclic Gly-(s-cis)Pro isosteres suitably protected for the Fmoc-based solid phase peptide synthesis. (R)-1-Phenylethylamine was introduced as chiral auxiliary to resolve racemic intermediates and obtain enantiopure compounds. Using methacrylamide as dipolarophile, the analogous Ala-Pro mimetics have been prepared in racemic form, whereas the same strategy applied to methyl itaconate failed to give the corresponding Asp-Pro mimetic.     

Air monitoring of volatile aromatic compounds by means of long-term exposure diffusive samplers

Results obtained by using "Analyst", a long-term diffusive sampler, in some monitoring campaigns, performed for the determination of benzene and the volatile aromatic pollutants, in five cities of the Italian Umbria Region and at the city of Forlì, are presented and discussed. First results of an inter comparison between the "Analyst" and "radial-type" diffusive samplers, carried out by the Regional Agency for the Ambient Protection (ARPA) of Forlì, point out substantial advantages with the long-time sampling devices.     

Synthesis of new fluorinated 1,5-benzoxazepine derivatives

A simple approach to the fluorinated 1,5-benzoxazepine ring system is described. By reacting commercially accessible aminophenols 1 and the trifluoroacetylvinyl ether 2, high yields of enaminones 3 were obtained. Functionalization of methyl group of compounds 3 gave rise to dieneamines 4 that were cyclized in acidic environment to benzoxazepine derivatives 5.     

1H detection in MAS solid-state NMR spectroscopy of biomacromolecules employing pulsed field gradients for residual solvent suppression

In this communication, we demonstrate the feasibility of 1H detection in MAS solid-state NMR for a microcrystalline, uniformly 2H,15N-labeled sample of a SH3 domain of chicken alpha-spectrin, using pulsed field gradients for suppression of water magnetization. Today, B0 gradients are employed routinely in solution-state NMR for coherence order selection and solvent suppression. We suggest to use gradients to purge water magnetization which cannot be suppressed using conventional water suppression schemes. The achievable gain in sensitivity for 1H detection is in the order of 5 compared to the 15N detected version of the experiment (at a MAS rotation frequency of 13.5 kHz). We expect that this labeling concept which achieves high sensitivity due to 1H detection, in combination with the possibility to measure long range 1H-1H distances as we have shown previously, to be a useful tool for the determination of protein structures in the solid state.     

Thermogravimetry as a screening tool for the estimation of the vapor pressures of pure compounds

A simplified approach was developed to estimate the vapor pressure of pure compounds from experimental data obtained by isothermal thermogravimetric (TG) analysis. A numerical procedure was developed to estimate the Antoine parameters of the substance by the analysis of isothermal TG data. The results of the experimental validations carried out evidenced that at least a preliminary estimation of vapour pressures of pure substances by the analysis of TG data is possible. The limited time and the reduced amounts of sample required for the experimental runs make the technique attractive with respect to the conventional and more accurate techniques for vapor pressure assessment.     

New 1,2,3‐triazolo[4,5‐e]1,2,4‐triazolo[4,3‐c]pyrimidine derivatives II

The 7‐chloro‐3‐(2‐chlorobenzyl)‐ and 7‐chloro‐3‐(2‐fluorobenzyl)‐1,2,3‐triazolo[4,5‐d]pyrimidines ( 1 and 4 ), by nucleophilic replacement with some hydrazides, gave the corresponding 7‐hydrazidoderivatives ( 2a‐e and 5a‐e ). These, by heating in Dowtherm, underwent an intramolecular cyclization to form the new tricyclic 7‐substituted‐3‐(2‐chlorobenzyl)‐ and 3‐(2‐fluorobenzyl)‐1,2,3‐triazolo[4,5‐e]1,2,4‐triazolo[4,3‐c]pyrimidines ( 3a‐d and 6a‐d ). The 7‐hydrazino‐3‐(2‐chlorobenzyl)‐ and 7‐hydrazino‐3‐(2‐fluorobenzyl)‐triazolo‐pyrimidines ( 9a and 9b ) were also prepared via the corresponding mercapto ( 7a and 7b ) and thiomethyl ( 8a and 8b ) derivatives.     

Identification of Dihydrolipoamide Dehydrogenase as Potential Target of Vemurafenib-Resistant Melanoma Cells

Background: Despite recent improvements in therapy, the five-year survival rate for patients with advanced melanoma is poor, mainly due to the development of drug resistance. The aim of the present study was to investigate the mechanisms underlying this phenomenon, applying proteomics and structural approaches to models of melanoma cells. Methods: Sublines from two human (A375 and SK-MEL-28) cells with acquired vemurafenib resistance were established, and their proteomic profiles when exposed to denaturation were identified through LC-MS/MS analysis. The pathways derived from bioinformatics analyses were validated by in silico and functional studies. Results: The proteomic profiles of resistant melanoma cells were compared to parental counterparts by taking into account protein folding/unfolding behaviors. Several proteins were found to be involved, with dihydrolipoamide dehydrogenase (DLD) being the only one similarly affected by denaturation in all resistant cell sublines compared to parental ones. DLD expression was observed to be increased in resistant cells by Western blot analysis. Protein modeling analyses of DLD’s catalytic site coupled to in vitro assays with CPI-613, a specific DLD inhibitor, highlighted the role of DLD enzymatic functions in the molecular mechanisms of BRAFi resistance. Conclusions: Our proteomic and structural investigations on resistant sublines indicate that DLD may represent a novel and potent target for overcoming vemurafenib resistance in melanoma cells.     

Environmentally Sustainable Achiral and Chiral Chromatographic Analysis of Amino Acids in Food Supplements

Two LC methods were developed for the achiral and chiral reversed-phase (RP) analysis of an amino acid (AA) pool in a food supplement, in compliance with the main paradigms of Green Chromatography. A direct achiral ion-pairing RP-HPLC method was optimized under gradient conditions with a water-ethanol (EtOH) eluent containing heptafluorobutyric acid (0.1%, v/v), to quantify the eight essential AAs (Ile, Leu, Lys, Met, Phe, Thr, Trp, and Val) contained in the food supplement. Thus, the usually employed acetonitrile was profitably substituted with the less toxic and more benign EtOH. The method was validated for Leu and Phe. The chiral LC method performed with a teicoplanin chiral stationary phase was developed with a water-EtOH (60:40, v/v) eluent with 0.1%, v/v acetic acid. The enantioselective analysis was carried out without any prior derivatization step. Both developed methods performed highly for all eight AAs and revealed that: (i) the content of six out of eight AAs was consistent with the manufacturer declaration; (ii) only L-AAs were present. Furthermore, it was demonstrated that a two-dimensional achiral–chiral configuration is possible in practice, making it even more environmentally sustainable. A molecular modelling investigation revealed interesting insights into the enantiorecognition mechanism of Lys.     

Investigation into the Use of Encorafenib to Develop Potential PROTACs Directed against BRAFV600E Protein

BRAF is a serine/threonine kinase frequently mutated in human cancers. BRAF^V600E mutated protein is targeted through the use of kinase inhibitors which are approved for the treatment of melanoma; however, their long-term efficacy is hampered by resistance mechanisms. The PROTAC-induced degradation of BRAF^V600E has been proposed as an alternative strategy to avoid the onset of resistance. In this study, we designed a series of compounds where the BRAF kinase inhibitor encorafenib was conjugated to pomalidomide through different linkers. The synthesized compounds maintained their ability to inhibit the kinase activity of mutated BRAF with IC₅₀ values in the 40–88 nM range. Selected compounds inhibited BRAF^V600E signaling and cellular proliferation of A375 and Colo205 tumor cell lines. Compounds 10 and 11, the most active of the series, were not able to induce degradation of mutated BRAF. Docking and molecular dynamic studies, conducted in comparison with the efficient BRAF degrader P5B, suggest that a different orientation of the linker bearing the pomalidomide substructure, together with a decreased mobility of the solvent-exposed part of the conjugates, could explain this behavior.     

Development of a Novel Microwave Distillation Technique for the Isolation of Cannabis sativa L. Essential Oil and Gas Chromatography Analyses for the Comprehensive Characterization of Terpenes and Terpenoids,Including Their Enantio-Distribution

A microwave distillation method was optimized for the extraction and isolation of cannabis essential oil from fresh and dried hemp inflorescences. The developed method enabled us to obtain a distilled product rich in terpenes and terpenoid compounds, responsible of the typical and unique smell of the cannabis plant. The distillate from different hemp cultivars, including Kompolti, Futura 75, Carmagnola, Felina 32 and Finola were characterized by using a gas chromatograph equipped with both mass spectrometer and flame ionization detectors. In a single chromatographic run, the identity and absolute amounts of distilled compounds were determined. Peak assignment was established using a reliable approach based on the usage of two identification parameters, named reverse match, and linear retention index filter. Absolute quantification (mg g⁻¹) of the analytes was performed using an internal standard method applying the flame ionization detector (FID) response factors according to each chemical family. An enantio-GC-MS method was also developed in order to evaluate the enantiomeric distribution of chiral compounds, an analytical approach commonly utilized for establishing the authenticity of suspicious samples.