Synthesis and Chemoinformatic Analysis of Fluorinated Piperidines as 3D Fragments for Fragment-Based Drug Discovery.

The concise synthesis of a small library of fluorinated piperidines from readily available dihydropyridinone derivatives has been described. The effect of the fluorination on different positions has then been evaluated by chemoinformatic tools. In particular, the compounds' pKa's have been calculated, revealing that the fluorine atoms notably lowered their basicity, which is correlated to the affinity for hERG channels resulting in cardiac toxicity. The "lead-likeness" and three-dimensionality have also been evaluated to assess their ability as useful fragments for drug design. A random screening on a panel of representative proteolytic enzymes was then carried out and revealed that one scaffold is recognized by the catalytic pocket of 3CLPro (main protease of SARS-CoV-2 coronavirus).

Accessing spiropiperidines from dihydropyridones through tandem triflation-allylation and ring-closing metathesis (RCM).

A novel approach to build 2-spiropiperidine moieties starting from dihydropyridones was developed. The triflic anhydride-promoted conjugate addition of allyltributylstannane onto dihydropyridones allowed for the formation of gem bis-alkenyl intermediates that were converted to the corresponding spirocarbocycles with excellent yields via ring closing metathesis. The vinyl triflate group generated on these 2-spiro-dihydropyridine intermediates could be successfully used as a chemical expansion vector for further transformations namely Pd-catalyzed cross-coupling reactions.

Structural and molecular bases to IRE1 activity modulation.

The Unfolded Protein response is an adaptive pathway triggered upon alteration of endoplasmic reticulum (ER) homeostasis. It is transduced by three major ER stress sensors, among which the Inositol Requiring Enzyme 1 (IRE1) is the most evolutionarily conserved. IRE1 is an ER-resident type I transmembrane protein exhibiting an ER luminal domain that senses the protein folding status and a catalytic kinase and RNase cytosolic domain. In recent years, IRE1 has emerged as a relevant therapeutic target in various diseases including degenerative, inflammatory and metabolic pathologies and cancer. As such several drugs altering IRE1 activity were developed that target either catalytic activity and showed some efficacy in preclinical pathological mouse models. In this review, we describe the different drugs identified to target IRE1 activity as well as their mode of action from a structural perspective, thereby identifying common and different modes of action. Based on this information we discuss on how new IRE1-targeting drugs could be developed that outperform the currently available molecules.

Structure Activity Relationship Studies around DB18, a Potent and Selective Inhibitor of CLK Kinases.

Three series of our lead CLK1 inhibitor DB18 have been designed, synthetized and tested against CLKs and DYRK1A kinases. Their cytotoxicity was subsequently measured on seven representative cancer cell lines. Guided by docking experiments, we focused on the less constrained part of the scaffold, and showed that drastically different substituents can be tolerated here. This work ended with the discovery of another promising derivative 12g, with IC50 = 0.004 µM in the inhibition of HsCLK1 and IC50 = 3.94 µM for the inhibition of HsDYRK1A. The SAR results are discussed in the light of extensive molecular modeling analyses. Finally, a kinome scan (463 human kinases) confirmed the outstanding selectivity of our lead compound DB18, suggesting that this scaffold is of prominent interest for selective CLK inhibitors. Altogether, these results pave the way for the development of inhibitors with novel selectivities in this family of kinases.

Gold-Catalyzed Synthesis of Substituted 3-Trifluoromethylpyrroles from Mesylated Amino Trifluoromethylpropargylic Alcohols.

A series of substituted 3-trifluoromethylpyrroles was obtained from trifluoromethylamino-ynol derivatives via a gold-catalyzed cyclization. Using fluorinated starting materials, after mesylation, allowed for the desired compounds to be obtained in good yields under mild conditions.

Isolation and Structure Identification of Novel Brominated Diketopiperazines from Nocardia ignorata-A Lichen-Associated Actinobacterium.

Actinobacteria are well known for their potential in biotechnology and their production of metabolites of interest. Lichens are a promising source of new bacterial strains, especially Actinobacteria, which afford a broad chemical diversity. In this context, the culture medium of the actinobacterium Nocardia ignorata, isolated from the terrestrial lichen Collema auriforme, was studied. The strain was cultivated in a BioFlo 115 bioreactor, and the culture medium was extracted using an XAD7HP resin. Five known diketopiperazines: cyclo (l-Pro-l-OMet) (1), cyclo (l-Pro-l-Tyr) (2), cyclo (d-Pro-l-Tyr) (3), cyclo (l-Pro-l-Val) (4), cyclo (l-Pro-l-Leu) (5), and one auxin derivative: indole-carboxaldehyde (8) were isolated, along with two new brominated diketopiperazines: cyclo (d-Pro-l-Br-Tyr) (6) and cyclo (l-Pro-l-Br-Tyr) (7). Structure elucidation was performed using HRMS and 1D and 2D NMR analysis, and the synthesis of compounds 6 and 7 was carried out in order to confirm their structure.

Integrating targeted gene expression and a skin model system to identify functional inhibitors of the UV activated p38 MAP kinase.

The stress-activated p38α MAP Kinase is an integral and critical component of the UV-induced inflammatory response. Despite the advances in recent years in the development of p38 kinase inhibitors, validation of these compounds in the diseased models remains limited. Based on the pharmacological profile of p38α inhibitor lead compound, SB203580, we synthesized a series of pyrrole-derivatives. Using UV-irradiated human skin punch-biopsies and cell cultures, we identified and validated the inhibitory activity of the derivatives by quantitatively measuring their effect on the expression of p38α target genes using real-time PCR. This approach not only identified pyrrole-2 as a unique derivative of this series that specifically inhibited the UV-activated p38α kinase, but also documented the skin permeation, bioavailability and reversible properties of this derivative in a 3D structure. The successful skin permeation of pyrrole-2 and its impact on AREG, COX-2 and MMP-9 gene expression demonstrates its potential use in modulating inflammatory processes in the skin. This study underscored the importance of using adapted biological models to identify accurate bioactive compounds.

Protolichesterinic acid derivatives: α-methylene-γ-lactones as potent dual activators of PPARγ and Nrf2 transcriptional factors.

PPARγ and Nrf2 are important transcriptional factors involved in many signaling pathways, especially in the anti-infectious response of macrophages. Compounds bearing a Michael acceptor moiety are well known to activate such transcriptional factors, we thus evaluated the potency of α,ß-unsaturated lactones synthesized using fluorous phase organic synthesis. Compounds were first screened for their cytotoxicity in order to select lactones for PPARγ and Nrf2 activation evaluation. Among them, two α-methylene-γ-lactones were identified as potent dual activators of PPARγ and Nrf2 in macrophages.

On the formation of a protic ionic liquid in nature.

The practical utility of ionic liquids (ILs) makes the absence (heretofore) of reported examples from nature quite puzzling, given the facility with which nature produces many other types of exotic but utilitarian substances. In that vein, we report here the identification and characterization of a naturally occurring protic IL. It can be formed during confrontations between the ants S. invicta and N. fulva. After being sprayed with alkaloid-based S. invicta venom, N. fulva detoxifies by grooming with its own venom, formic acid. The mixture is a viscous liquid manifestly different from either of the constituents. Further, we find that the change results as a consequence of formic acid protonation of the N centers of the S. invicta venom alkaloids. The resulting mixed-cation ammonium formate milieu has properties consistent with its classification as a protic IL.

UV-protectant metabolites from lichens and their symbiotic partners.

Lichens are structurally complex symbiotic organisms that are exposed to a wide variety of external conditions (extreme temperatures, desiccation, UV radiation, etc.). These poikilohydric organisms have developed various mechanisms of photoprotection, such as light scattering, radiation screening, thermal dissipation, activation of antioxidant defense and macromolecules and membrane repair. These unique organisms produce a vast array of compounds, with more than 1000 secondary metabolites known. An important protective mechanism of lichens is the production of UV screening compounds, such as phenolic compounds (depsidones, depsides, diphenyl ethers), anthraquinones, xanthones or shikimic acid derivatives (calycin, mycosporines, scytonemin). Due to the harmful effects of the UVA wavelengths of sunlight, the search for new sunscreens remains important. We herein propose a review that focuses on the UV protectants from lichens and their symbiotic partners (lichenized fungi, green alga, cyanobacteria). In fact, lichens produce unique and/or efficient UV filters such as depsidones (lobaric acid, pannarin, etc.), depsides (atranorin, gyrophoric acid, etc.), diphenyl ethers (epiphorellic acids, buellin), bisxanthones (secalonic acids, etc.), mycosporines and MAAs, scytonemin along with classical pigments (melanin, carotenoids). We propose to classify these compounds with regard to their chemical structures and review the physicochemical properties that act as UV filters. While the most abundant lichen polyfunctionalized aromatic compounds, belonging to orsellinic derivatives, are UVB screens, these organisms produce strong UVA filters, e.g., calycin (pulvinic acid derivatives), bisxanthones (secalonic acids), scytonemin or mycosporines and MAAs with the latter ones exhibiting attractive properties as photoprotectants.

Gold-mediated synthesis and functionalization of chiral halopyridones.

A rapid and efficient one-step halopyridone synthesis has been developed based on gold-catalyzed cyclization of ß-amino-ynone intermediates and halodeauration process.

Total synthesis of (-)-epimyrtine by a gold-catalyzed hydroamination approach.

A new approach to the total synthesis of (-)-epimyrtine has been developed from D-alanine. The key step to access the enantiopure pyridone intermediate was achieved by a gold-mediated cyclization. Finally, various transformations afforded the natural product in a few steps and good overall yield.

Total synthesis of dendrobate alkaloid (+)-241D, isosolenopsin and isosolenopsin A: application of a gold-catalyzed cyclization.

A new approach to total syntheses of piperidine alkaloids (+)-241D, isosolenopsin and isosolenopsin A has been developed from D-alanine. The key step to access the chiral pyridinone intermediate was achieved via a gold mediated cyclization. Finally, various reduction conditions afforded the natural products in few steps and good overall yields.

Prodrugs as new therapies against Chagas disease: in vivo synergy between Trypanosoma cruzi proline racemase inhibitors and benznidazole.

OBJECTIVES: Chagas disease, caused by the parasitic protozoan Trypanosoma cruzi, affects approximately 6-7 million people worldwide. There are limited available therapies and they exhibit low efficacy, often high toxicity in chronic cases and some drug resistance. In this study, our objective was to develop ester prodrugs that inhibit proline racemase (TcPRAC), a parasitic enzyme previously identified and characterised as a promising target because of its essential role in the parasite's life cycle and virulence, and to test their activity against T. cruzi. METHODS: Using structural bioinformatics, we modelled several functional intermediates of the catalytic site between the opened and closed conformations of TcPRAC based on its crystal structures in complex with its competitive inhibitor, pyrrole-2-carboxylic acid. Guided by these intermediates, which were later validated in cocrystals, we designed and evaluated numerous compounds and tested them enzymatically on live parasites and in mice with our quick and straightforward drug screening method, which is based on state-of-the-art bioluminescent T. cruzi parasites injected subcutaneously. RESULTS: Some of our novel compounds specifically inhibited racemase activity, as determined through biochemical assays, and covalently bound to TcPRAC. Furthermore, the corresponding ester prodrugs were effective in killing parasites in vitro. Bioluminescent T. cruzi assays in mice showed that JR1531, a TcPRAC inhibitor prodrug, can kill parasites in living animals, with boosted action when combined with low doses of benznidazole. CONCLUSION: This approach, based on TcPRAC inhibitor prodrugs in association with low doses of benznidazole, may lead to more effective, specific and non-toxic therapies against Chagas disease.

Irreversible inhibitors of the proline racemase unveil innovative mechanism of action as antibacterial agents against Clostridioides difficile.

Proline racemases (PRAC), catalyzing the l-proline and d-proline interconversion, are essential factors in eukaryotic pathogens such as Trypanosoma cruzi, Trypanosoma vivax, and Clostridioides difficile. If the discovery of irreversible inhibitors of T. cruzi PRAC (TcPRAC) led to innovative therapy of the Chagas disease, no inhibitors of CdPRAC have been discovered to date. However, C. difficile, due to an increased incidence in recent years, is considered as a major cause of health threat. In this work, we have taken into account the similarity between TcPRAC and CdPRAC enzymes to design new inhibitors of CdPRAC. Starting from (E) 4-oxopent-2-enoic acid TcPRAC irreversible inhibitors, we synthesized 4-aryl substituted analogs and evaluated their CdPRAC enzymatic inhibition against eleven strains of C. difficile. This study resulted in promising candidates and allowed for identification of (E)-4-(3-bromothiophen-2-yl)-4-oxobut-2-enoic acid 20 that was chosen for complementary in vivo studies and did not reveal in vivo toxicity.

Synthesis of substituted pyrrolin-4-ones from amino acids in mild conditions via a gold-catalyzed approach.

The gold-catalyzed cyclization of various alpha-amino-ynone derivatives gave the corresponding pyrrolin-4-ones in high yields. Moreover, the use of gold(III) oxide as catalyst allows a moderate to total stereocontrol during the cyclization. These pyrrolin-4-ones are highly useful intermediates for the synthesis of functionalized pyrrolidines and other natural products.

Lichen butyrolactone derivatives disrupt oral bacterial membrane.

We have previously demonstrated that out of the butyrolactones series synthesized based on the natural lichen metabolite lichesterinic acid, compound (B-13) was the most effective against oral bacteria. However, its antibacterial mechanism is still unknown. In this study, we have investigated its bacterial localization by synthesizing a fluorescently labeled B-13 with NBD while maintaining its antibacterial activity. We showed that this compound binds to Streptococcus gordonii cell surface, as demonstrated by HPLC analysis. By adhering to cell surface, B-13 induced cell wall disruption leading to the release of bacterial constituents and consequently, the death of S. gordonii, a Gram-positive bacterium. A Gram-negative counterpart, Porphyromanas gingivalis, showed also cracked and ruptured cells in the presence of B-13. Besides, we also demonstrated that the analog of B-13, B-12, has also induced disruption of P. gingivalis and S. gordonii. This study revealed that butyrolactones can be considered as potent antibacterial compounds against oral pathogens causing medical complications.

Designed mono- and di-covalent inhibitors trap modeled functional motions for Trypanosoma cruzi proline racemase in crystallography.

Chagas disease, caused by Trypanosoma cruzi, affects millions of people in South America and no satisfactory therapy exists, especially for its life threatening chronic phase. We targeted the Proline Racemase of T. cruzi, which is present in all stages of the parasite life cycle, to discover new inhibitors against this disease. The first published crystal structures of the enzyme revealed that the catalytic site is too small to allow any relevant drug design. In previous work, to break through the chemical space afforded to virtual screening and drug design, we generated intermediate models between the open (ligand free) and closed (ligand bound) forms of the enzyme. In the present work, we co-crystallized the enzyme with the selected inhibitors and found that they were covalently bound to the catalytic cysteine residues in the active site, thus explaining why these compounds act as irreversible inhibitors. These results led us to the design of a novel, more potent specific inhibitor, NG-P27. Co-crystallization of this new inhibitor with the enzyme allowed us to confirm the predicted protein functional motions and further characterize the chemical mechanism. Hence, the catalytic Cys300 sulfur atom of the enzyme attacks the C2 carbon of the inhibitor in a coupled, regiospecific-stereospecific Michael reaction with trans-addition of a proton on the C3 carbon. Strikingly, the six different conformations of the catalytic site in the crystal structures reported in this work had key similarities to our intermediate models previously generated by inference of the protein functional motions. These crystal structures span a conformational interval covering roughly the first quarter of the opening mechanism, demonstrating the relevance of modeling approaches to break through chemical space in drug design.

Solid-phase synthesis and evaluation of libraries of substituted 4,5-dihydropyridazinones as vasodilator agents.

The solid-phase parallel preparation of a library of 4,5-dihydropyridazin-3(2H)-one derivatives substituted at position 6 with piperazinylmethyl or tetrahydroquinolinylmethyl groups and analogues (3) is reported. Polymer-supported gamma-keto-delta-aminoesters prepared from Wang resin reacted with hydrazine or methylhydrazine to afford pyridazinones in good yields after a cyclization cleavage approach. We have evaluated these novel analogues and several compounds of other series (1, 2) for their vasorelaxant effect. Among the products tested, 3l and 3d proved to be efficacious and potent relaxant agents of the isolated rat aorta. Inhibitors of phosphodiesterase (PDE3), responsible for the breakdown of cyclic AMP in the vascular smooth muscle, are currently developed for cardiac heart failure because of their inotropic effect and coronary vasodilatation. We had expected that the vasodilatation induced by 3l, as efficient as reference PDE3 inhibitors, milrinone or CI-930, to be due to PDE3 inhibition. However 3l and 3d exhibited a low inhibitory effect against PDE3 isoenzyme activity. These compounds induced a significant vasorelaxation, which could be of therapeutic interest even if their mechanism of action remains to be determined.

Combined approaches for drug design points the way to novel proline racemase inhibitor candidates to fight Chagas' disease.

Chagas' disease is caused by Trypanosoma cruzi, a protozoan transmitted to humans by blood-feeding insects, blood transfusion or congenitally. Previous research led us to discover a parasite proline racemase (TcPRAC) and to establish its validity as a target for the design of new chemotherapies against the disease, including its chronic form. A known inhibitor of proline racemases, 2-pyrrolecarboxylic acid (PYC), is water-insoluble. We synthesized soluble pyrazole derivatives, but they proved weak or inactive TcPRAC inhibitors. TcPRAC catalytic site is too small and constrained when bound to PYC to allow efficient search for new inhibitors by virtual screening. Forty-nine intermediate conformations between the opened enzyme structure and the closed liganded one were built by calculating a transition path with a method we developed. A wider range of chemical compounds could dock in the partially opened intermediate active site models in silico. Four models were selected for known substrates and weak inhibitors could dock in them and were used to screen chemical libraries. Two identified soluble compounds, (E)-4-oxopent-2-enoic acid (OxoPA) and its derivative (E)-5-bromo-4-oxopent-2-enoic acid (Br-OxoPA), are irreversible competitive inhibitors that presented stronger activity than PYC on TcPRAC. We show here that increasing doses of OxoPA and Br-OxoPA hamper T. cruzi intracellular differentiation and fate in mammalian host cells. Our data confirm that through to their binding mode, these molecules are interesting and promising as lead compounds for the development of chemotherapies against diseases where active proline racemases play essential roles.