Different chemical proteomic approaches to identify the targets of lapatinib.

The process of identifying the protein targets and off-targets of a biologically active compound is of great importance in modern drug discovery. Various chemical proteomics approaches have been established for this purpose. To compare the different approaches, and to understand which method would provide the best results, we have chosen the EGFR inhibitor lapatinib as an example molecule. Lapatinib derivatives were designed using linkers with motifs, including amino (amidation), alkyne (click chemistry) and the diazirine group (photo-affinity). These modified lapatinib analogues were validated for their ability to inhibit EGFR activity in vitro and were shown to pull down purified recombinant EGFR protein. In all of the approaches evaluated here, we identified EGFR as the main protein target from the lysate of immortalised cell line expressing EGFR, thus validating its potential use to identify unknown protein targets. Taken together, the results reported here give insight into the cellular activities of lapatinib.

Investigation of Praziquantel/Cyclodextrin Inclusion Complexation by NMR and LC-HRMS/MS: Mechanism, Solubility, Chemical Stability, and Degradation Products.

Praziquantel (PZQ) is a biopharmaceutical classification system (BCS) class II anthelmintic drug characterized by poor solubility and a bitter taste, both of which can be addressed by inclusion complexation with cyclodextrins (CD). In this work, a comprehensive investigation of praziquantel/cyclodextrin (PZQ/CD) complexes was conducted by means of UV-vis spectroscopy, spectrofluorimetry, NMR spectroscopy, liquid chromatography-high-resolution mass spectrometry (LC-HRMS/MS), and molecular modeling. Phase solubility studies revealed that among four CDs tested, the randomly methylated ß-CD (RMßCD) and the sulfobutylether sodium salt ß-CD (SBEßCD) resulted in the highest increase in PZQ solubility (approximately 16-fold). The formation of 1:1 inclusion complexes was confirmed by HRMS, NMR, and molecular modeling. Both cyclohexane and the central pyrazino ring, as well as an aromatic part of PZQ are included in the CD central cavity through several different binding modes, which exist simultaneously. Furthermore, the influence of CDs on PZQ stability was investigated in solution (HCl, NaOH, H2O2) and in the solid state (accelerated degradation, photostability) by ultra-high-performance liquid chromatography-diode array detection-tandem mass spectrometry (UPLC-DAD/MS). CD complexation promoted new degradation pathways of the drug. In addition to three already known PZQ degradants, seven new degradation products were identified (m/z 148, 215, 217, 301, 327, 343, and 378) and their structures were proposed based on HRMS/MS data. Solid complexes were prepared by mechanochemical activation, a solvent-free and ecologically acceptable method.

Degradation products of azetidine core G334089 - Isolation, structure elucidation and pathway.

An extensive forced degradation study using hydrolytic degradation conditions was performed on G334089, the S-enantiomer of the free fatty acid receptor 2 (FFA2) antagonist GLPG0974, to identify the degradation product structures and discern degradation pathways. Not all degradation products generated ions in the MS spectra, while several others were isomers, so more rigorous degradation conditions were applied to increase the degradant yield. Esterification of the degradants facilitated isolation via preparative HPLC and subsequent NMR and MS characterisation. The determined structures, retention times and fragmentation patterns were used to identify the original degradation products and postulate a degradation pathway. In addition to the expected amide bond hydrolysis, a second degradation mechanism involving azetidine activation through formation of an azetidinium ion was demonstrated.

Benzoxaboroles-Novel Autotaxin Inhibitors.

Autotaxin (ATX) is an extracellular enzyme that hydrolyses lysophosphatidylcholine (LPC) to lysophosphatidic acid (LPA), which has a role in the mediation of inflammation, fibrosis and cancer. ATX is a drug target that has been the focus of many research groups during the last ten years. To date, only one molecule, Ziritaxestat (GLPG1690) has entered the clinic; it is currently in Phase 3 clinical trials for idiopathic pulmonary fibrosis. Other small molecules, with different binding modes, have been investigated as ATX inhibitors for cancer including compounds possessing a boronic acid motif such as HA155. In this work, we targeted new, improved inhibitors of ATX that mimic the important interactions of boronic acid using a benzoxaborole motif as the acidic warhead. Furthermore, we aimed to improve the plasma stability of the new compounds by using a more stable core spacer than that embedded in HA155. Compounds were synthesized, evaluated for their ATX inhibitory activity and ADME properties in vitro, culminating in a new benzoxaborole compound, 37, which retains the ATX inhibition activity of HA155 but has improved ADME properties (plasma protein binding, good kinetic solubility and rat/human plasma stability).

Around the macrolide - Impact of 3D structure of macrocycles on lipophilicity and cellular accumulation.

The aim of this study was to investigate lipophilicity and cellular accumulation of rationally designed azithromycin and clarithromycin derivatives at the molecular level. The effect of substitution site and substituent properties on a global physico-chemical profile and cellular accumulation of investigated compounds was studied using calculated structural parameters as well as experimentally determined lipophilicity. In silico models based on the 3D structure of molecules were generated to investigate conformational effect on studied properties and to enable prediction of lipophilicity and cellular accumulation for this class of molecules based on non-empirical parameters. The applicability of developed models was explored on a validation and test sets and compared with previously developed empirical models.

Structure and conformational analysis of spiroketals from 6-O-methyl-9(E)-hydroxyiminoerythronolide A.

Three novel spiroketals were prepared by a one-pot transformation of 6-O-methyl-9(E)-hydroxyiminoerythronolide A. We present the formation of a [4.5]spiroketal moiety within the macrolide lactone ring, but also the unexpected formation of a 10-C=11-C double bond and spontaneous change of stereochemistry at position 8-C. As a result, a thermodynamically stable structure was obtained. The structures of two new diastereomeric, unsaturated spiroketals, their configurations and conformations, were determined by means of NMR spectroscopy and molecular modelling. The reaction kinetics and mechanistic aspects of this transformation are discussed. These rearrangements provide a facile synthesis of novel macrolide scaffolds.

Determination of aqueous stability and degradation products of series of coumarin dimers.

The stability in aqueous solution of five classes of coumarin dimers (I-V, compounds 1-29) was studied by HPLC-MS/MS at various pH values. The relationship between chemical structure and stability is discussed. It was found that dimeric compounds with strong electron withdrawing groups (EWGs) on the α-carbon to the bridging C-atom are stable at all pH values, whereas other derivatives undergo retro-Michael addition at rates which are also affected by the substituents on the aromatic rings. In some cases formation of stable isomers or oxidation products was observed. In order to evaluate their developability and potential for progression to in vivo studies, representative compounds were tested in an in vitro microsomal stability assay.

Identification of impurities in acarbose by using an integrated liquid chromatography-nuclear magnetic resonance and liquid chromatography-mass spectrometry approach.

The usefulness of applying an integrated LC-NMR and LC-MS approach to acarbose bulk drug impurity profiling is demonstrated. LC-MS and LC-NMR methodologies were employed for the online separation and structural elucidation of a final drug product. Combining data provided by the stop-flow LC-NMR and LC-MS experiments made it possible to identify the main components present in the acarbose sample. Spectral analysis revealed that A and B were known impurities while C was an unknown compound. LC-MS and LC-NMR analyses revealed that C was a pentasaccharide differing from the acarbose in number and nature of sugar subunits in the molecule. It was subsequently isolated and its structure was confirmed by the offline 1- and 2-D NMR experiments, and atom assignment was made.

Combined use of liquid chromatography-nuclear magnetic resonance spectroscopy and liquid chromatography-mass spectrometry for the characterization of an acarbose degradation product.

Directly coupled LC-MS and LC-NMR were applied to identify and structurally characterize an acarbose degradation product A in acidic media. A comparative analysis of the stop-flow LC-NMR (1H and TOCSY) and LC-MS data provided evidence that A is structurally related to acarbose, differing from the parent compound in a number of subunits present in the molecule. Spectral analysis revealed that A was the alpha-glucosidase inhibitor amylostatin XG. Complementary information obtained from the two methods led to the structural elucidation of A which was later corroborated by high-resolution NMR spectroscopy of the isolated molecule.