Identification of Riluzole derivatives as novel calmodulin inhibitors with neuroprotective activity by a joint synthesis, biosensor, and computational guided strategy.

The development of new molecules for the treatment of calmodulin related cardiovascular or neurodegenerative diseases is an interesting goal. In this work, we introduce a novel strategy with four main steps: (1) chemical synthesis of target molecules, (2) Förster Resonance Energy Transfer (FRET) biosensor development and in vitro biological assay of new derivatives, (3) Cheminformatics models development and in vivo activity prediction, and (4) Docking studies. This strategy is illustrated with a case study. Firstly, a series of 4-substituted Riluzole derivatives 1-3 were synthetized through a strategy that involves the construction of the 4-bromoriluzole framework and its further functionalization via palladium catalysis or organolithium chemistry. Next, a FRET biosensor for monitoring Ca2+-dependent CaM-ligands interactions has been developed and used for the in vitro assay of Riluzole derivatives. In particular, the best inhibition (80%) was observed for 4-methoxyphenylriluzole 2b. Besides, we trained and validated a new Networks Invariant, Information Fusion, Perturbation Theory, and Machine Learning (NIFPTML) model for predicting probability profiles of in vivo biological activity parameters in different regions of the brain. Next, we used this model to predict the in vivo activity of the compounds experimentally studied in vitro. Last, docking study conducted on Riluzole and its derivatives has provided valuable insights into their binding conformations with the target protein, involving calmodulin and the SK4 channel. This new combined strategy may be useful to reduce assay costs (animals, materials, time, and human resources) in the drug discovery process of calmodulin inhibitors.

Prediction of Antileishmanial Compounds: General Model, Preparation, and Evaluation of 2-Acylpyrrole Derivatives.

In this work, the SOFT.PTML tool has been used to pre-process a ChEMBL dataset of pre-clinical assays of antileishmanial compound candidates. A comparative study of different ML algorithms, such as logistic regression (LOGR), support vector machine (SVM), and random forests (RF), has shown that the IFPTML-LOGR model presents excellent values of specificity and sensitivity (81-98%) in training and validation series. The use of this software has been illustrated with a practical case study focused on a series of 28 derivatives of 2-acylpyrroles 5a,b, obtained through a Pd(II)-catalyzed C-H radical acylation of pyrroles. Their in vitro leishmanicidal activity against visceral (L. donovani) and cutaneous (L. amazonensis) leishmaniasis was evaluated finding that compounds 5bc (IC50 = 30.87 µM, SI > 10.17) and 5bd (IC50 = 16.87 µM, SI > 10.67) were approximately 6-fold more selective than the drug of reference (miltefosine) in in vitro assays against L. amazonensis promastigotes. In addition, most of the compounds showed low cytotoxicity, CC50 > 100 µg/mL in J774 cells. Interestingly, the IFPMTL-LOGR model predicts correctly the relative biological activity of these series of acylpyrroles. A computational high-throughput screening (cHTS) study of 2-acylpyrroles 5a,b has been performed calculating >20,700 activity scores vs a large space of 647 assays involving multiple Leishmania species, cell lines, and potential target proteins. Overall, the study demonstrates that the SOFT.PTML all-in-one strategy is useful to obtain IFPTML models in a friendly interface making the work easier and faster than before. The present work also points to 2-acylpyrroles as new lead compounds worthy of further optimization as antileishmanial hits.

Catalytic cyclopropanation reactions with α-silyl-, germanyl- and stannyl carbenes generated from cyclopropenes.

Silylcyclopropenes are employed as precursors of α-silyl vinyl carbenes and trapped with alkenes. Cyclopropylsilanes were obtained in good yields with ample scope and complete regio- and diastereoselectivity. Stereoretentive protodesilylations enabled access to cis-1,2-disubstituted cyclopropanes. Cyclopropylstannanes and -germanes can also be prepared from the corresponding cyclopropenes.

Pd(II)-Catalyzed Fujiwara-Moritani Reactions for the Synthesis and Functionalization of Substituted Coumarins.

Highly substituted coumarins, privileged and versatile scaffolds for bioactive natural products and fluorescence imaging, are obtained via a Pd(II)-catalyzed direct C-H alkenylation reaction (Fujiwara-Moritani reaction), which has emerged as a powerful tool for the construction and functionalization of heterocyclic compounds because of its chemical versatility and its environmental advantages. Thus, a selective 6-endo cyclization led to 4-substituted coumarins in moderate yields. Selected examples have been further functionalized in C3 through a second intermolecular C-H alkenylation reaction to give coumarin-acrylate hybrids, whose fluorescence spectra have been measured.

Palladium-mediated synthesis and biological evaluation of C-10b substituted Dihydropyrrolo[1,2-b]isoquinolines as antileishmanial agents.

The development of new molecules for the treatment of leishmaniasis is, a neglected parasitic disease, is urgent as current anti-leishmanial therapeutics are hampered by drug toxicity and resistance. The pyrrolo[1,2-b]isoquinoline core was selected as starting point, and palladium-catalyzed Heck-initiated cascade reactions were developed for the synthesis of a series of C-10 substituted derivatives. Their in vitro leishmanicidal activity against visceral (L. donovani) and cutaneous (L. amazonensis) leishmaniasis was evaluated. The best activity was found, in general, for the 10-arylmethyl substituted pyrroloisoquinolines. In particular, 2ad (IC50 = 3.30 µM, SI > 77.01) and 2bb (IC50 = 3.93 µM, SI > 58.77) were approximately 10-fold more potent and selective than the drug of reference (miltefosine), against L. amazonensis on in vitro promastigote assays, while 2ae was the more active compound in the in vitro amastigote assays (IC50 = 33.59 µM, SI > 8.93). Notably, almost all compounds showed low cytotoxicity, CC50 > 100 µg/mL in J774 cells, highest tested dose. In addition, we have developed the first Perturbation Theory Machine Learning (PTML) algorithm able to predict simultaneously multiple biological activity parameters (IC50, Ki, etc.) vs. any Leishmania species and target protein, with high values of specificity (>98%) and sensitivity (>90%) in both training and validation series. Therefore, this model may be useful to reduce time and assay costs (material and human resources) in the drug discovery process.

Molecular docking, SAR analysis and biophysical approaches in the study of the antibacterial activity of ceramides isolated from Cissus incisa.

The developing of antibacterial resistance is becoming in crisis. In this sense, natural products play a fundamental role in the discovery of antibacterial agents with diverse mechanisms of action. Phytochemical investigation of Cissus incisa leaves led to isolation and characterization of the ceramides mixture (1): (8E)-2-(tritriacont-9-enoyl amino)-1,3,4-octadecanetriol-8-ene (1-I); (8E)-2-(2',3'-dihydroxyoctacosanoyl amino)-1,3,4-octadecanetriol-8-ene (1-II); (8E)-2-(2'-hydroxyheptacosanoyl amino)-1,3,4-octadecanetriol-8-ene (1-III); and (8E)-2-(-2'-hydroxynonacosanoyl amino)-1,3,4-octadecanetriol-8-ene (1-IV). Until now, this is the first report of the ceramides (1-I), (1-II), and (1-IV). The structures were elucidated using NMR and mass spectrometry analyses. Antibacterial activity of ceramides (1) and acetylated derivates (2) was evaluated against nine multidrug-resistant bacteria by Microdilution method. (1) showed the best results against Gram-negatives, mainly against carbapenems-resistant Acinetobacter baumannii with MIC = 50 µg/mL. Structure-activity analysis and molecular docking revealed interactions between plant ceramides with membrane proteins, and enzymes associated with biological membranes of Gram-negative bacteria, through hydrogen bonding of functional groups. Vesicular contents release assay showed the capacity of (1) to disturb membrane permeability detected by an increase of fluorescence probe over time. The membrane disruption is not caused for ceramides lytic action on cell membranes, according in vitro hemolyticactivity results. Combining SAR analysis, bioinformatics and biophysical techniques, and also experimental tests, it was possible to explain the antibacterial action of these natural ceramides.

Carbopalladation/Suzuki Coupling Cascade for the Generation of Quaternary Centers: Access to Pyrrolo[1,2-b]isoquinolines.

A convergent route to pyrrolo[1,2-b]isoquinolines with a quaternary center at C-10 has been developed, which implies a sequential Pd(0)-catalyzed carbopalladation followed by cross-coupling reaction with boronic acids. The adequate catalytic system and experimental conditions, with and without the use of phosphane ligands, have been selected to control the chemoselectivity of the process, allowing a 6-exo-carbopalladation to generate a quaternary center and avoiding a direct Suzuki coupling. A variety of electron-rich and electron-deficient arylboronic acids can be used providing an efficient route to substituted pyrrolo[1,2-b]isoquinolines in moderate to good yields (up to 94%, 22 examples).

A simple and reproducible method for quantification of human tear lipids with ultrahigh-performance liquid chromatography-mass spectrometry.

Purpose: The purpose was to select a simple and reproducible method for lipid measurements of human tears with ultrahigh-performance liquid chromatography-mass spectrometry (UHPLC-MS). Two sample preparation procedures were evaluated and compared: the Bligh and Dyer (BD) liquid-liquid extraction method with chloroform and methanol and protein precipitation with isopropanol (IPA). Methods: Reproducibility and recovery efficiencies of 20 non-endogenous internal lipid standards were tested in 10-µl tear samples from healthy subjects. The lipid coverage and the simplicity of execution were also assessed. Lipid profiles of the tear extracts were acquired with UHPLC-MS, uhpland the lipids were identified using SimLipid software. Results: Both methods were robust producing good lipid coverage and reproducibility and high recovery efficiencies. The two protocols identified a 69-feature tear lipidome that covered 11 lipid classes from six different lipid categories. The main differences in recovery were due to the intrinsic lipid selectivity of each solvent. Although both methods were similarly efficient in recovering O-acyl-ω-hydroxy fatty acid (OAHFAs) and non-polar lipids, polar lipids were more efficiently recovered with IPA precipitation, which, in turn, exhibited higher reproducibility. In addition, IPA precipitation is automatable and simpler than the BD approach. Conclusions: IPA precipitation is an excellent procedure for extracting lipids from small tear volumes for quantitative large-scale, untargeted lipid profiling, which may be useful for identifying lipid biomarkers in tears from patients with different ocular surface pathologies, allowing personalized therapies to be designed.