Design, synthesis and bioactivity evaluation of 4-hydroxycoumarin derivatives as potential anti-inflammatory agents against acute lung injury and colitis.

Acute lung injury (ALI) and inflammatory bowel disease (IBD) are common inflammatory illnesses that seriously affect people's health. Herein, a series of 4-hydroxylcoumarin (4-HC) derivatives were designed and synthesized. The inhibitory effects of these compounds on LPS-induced interleukin-6 (IL-6) release from J774A.1 cells were then screened via ELISA assay, compound B8 showed 3 times more active than the lead compound 4-HC. The most active compound B8 had the IC50 values of 4.57 µM and 6.51 µM for IL-6 release on mouse cells J774A.1 and human cells THP-1, respectively. Furthermore, we also found that B8 could act on the MAPK pathway. Based on the target prediction results of computer virtual docking, kinase inhibitory assay was carried out, and it revealed that targeting IRAK1 was a key mechanism for B8 to exert anti-inflammatory activity. Moreover, B8 exerted a good therapeutic effect on the dextran sulfate sodium (DSS)-induced colitis model and liposaccharide (LPS)-induced ALI mouse models. The acute toxicity experiments indicated that high-dose B8 caused no adverse reactions in mice, confirming its safety in vivo. Additionally, the preliminary pharmacokinetic (PK) parameters of B8 in SD rats were also examined, revealing a bioavailability (F) of 28.72 %. In conclusion, B8 is a potential candidate of drug for the treatment of ALI and colitis.

Fishing antioxidant 4-hydroxycoumarin derivatives: synthesis, characterization, and in vitro assessments.

Coumarins represent a diverse class of natural compounds whose importance in pharmaceutical and agri-food sectors has motivated multiple novel synthetic derivatives with broad applicability. The phenolic moiety in 4-hydroxycoumarins underscores their potential to modulate the equilibrium between free radicals and antioxidant species within biological systems. The aim of this work was to assess the antioxidant activity of 18 4-hydroxycoumarin coumarin derivatives, six of which are commercially available and the other 12 were synthesized and chemically characterized and described herein. The 4-hydroxycoumarins were prepared by a two steps synthetic strategy with satisfactory yields. Their antioxidant potential was evaluated through three in vitro methods, two free radical-scavenging assays (DPPH• and ABTS•+) and a metal chelating activity assay. Six synthetic coumarins (4a, 4g, 4h, 4i, 4k, 4l) had a scavenging capacity of DPPH• higher than butylated hydroxytoluene (BHT) (IC50 = 0.58 mmol/L) and compound 4a (4-hydroxy-6-methoxy-2 H-chromen-2-one) with an IC50 = 0.05 mmol/L outperformed both BHT and ascorbic acid (IC50 = 0.06 mmol/L). Nine hydroxycoumarins had a scavenging capacity against ABTS•+ greater (C3, 4a, 4c) or comparable (C1, C2, C4, C6, 4g, 4l) to Trolox (IC50 = 34.34 µmol/L). Meanwhile, the set had a modest ferrous chelation capacity, but most of them (C2, C5, C6, 4a, 4b, 4h, 4i, 4j, 4k, 4l) reached up to more than 20% chelating ability percentage. Collectively, this research work provides valuable structural insights that may determine the scavenging and metal chelating activity of 4-hydroxycoumarins. Notably, substitutions at the C6 position appeared to enhance scavenging potential, while the introduction of electron-withdrawing groups showed promise in augmenting chelation efficiency.

Synthesis, Crystallographic, Quantum Chemical, Antitumor, and Molecular Docking/Dynamic Studies of 4-Hydroxycoumarin-Neurotransmitter Derivatives.

In this contribution, four new compounds synthesized from 4-hydroxycoumarin and tyramine/octopamine/norepinephrine/3-methoxytyramine are characterized spectroscopically (IR and NMR), chromatographically (UHPLC-DAD), and structurally at the B3LYP/6-311++G*(d,p) level of theory. The crystal structure of the 4-hydroxycoumarin-octopamine derivative was solved and used as a starting geometry for structural optimization. Along with the previously obtained 4-hydroxycoumarin-dopamine derivative, the intramolecular interactions governing the stability of these compounds were quantified by NBO and QTAIM analyses. Condensed Fukui functions and the HOMO-LUMO gap were calculated and correlated with the number and position of OH groups in the structures. In vitro cytotoxicity experiments were performed to elucidate the possible antitumor activity of the tested substances. For this purpose, four cell lines were selected, namely human colon cancer (HCT-116), human adenocarcinoma (HeLa), human breast cancer (MDA-MB-231), and healthy human lung fibroblast (MRC-5) lines. A significant selectivity towards colorectal carcinoma cells was observed. Molecular docking and molecular dynamics studies with carbonic anhydrase, a prognostic factor in several cancers, complemented the experimental results. The calculated MD binding energies coincided well with the experimental activity, and indicated 4-hydroxycoumarin-dopamine and 4-hydroxycoumarin-3-methoxytyramine as the most active compounds. The ecotoxicology assessment proved that the obtained compounds have a low impact on the daphnia, fish, and green algae population.

Comparative pharmacokinetics of difethialone stereoisomers in male and female rats and mice: development of an intra- and inter-species model to predict the suitable formulation mix.

The ecotoxicity of anticoagulants used for rodent pests' management is a major concern, particularly with second generation anticoagulants, which are more persistent in the body of rodents and therefore more likely to cause secondary exposure in their predators. One of the solutions envisaged to mitigate this risk is to use stereoisomers of these anticoagulants, each of which has particular pharmacokinetics. However, the few studies published to date have considered only one species and one sex. Here, we study the pharmacokinetics of the 4 stereoisomers of 3.4 mg/kg of difethialone in rats (Rattus norvegicus) and 3 mg/kg in mice (Mus musculus) in both sexes and propose a model to choose the optimal stereoisomer efficacy/ecotoxicity mixture for the management of all these animals. Our results show that while the most persistent stereoisomer (E3-cis) is common to both species and sexes, the pharmacokinetics of the other stereoisomers show marked differences between sexes and species. Thus, the area under curve (AUC) of E4-trans in male rats is four times lower than in females or mice, making it a priori unusable in male rats. Conversely, our modeling seems to show that the E1-trans stereoisomer seems to offer the best compromise AUC persistence. In conclusion, we highlight that studies on anticoagulants must necessarily integrate research on the effect of gender and species both on efficacy and with regard to the ecotoxicity of these molecules.

Conformational Flexibility of A Highly Conserved Helix Controls Cryptic Pocket Formation in FtsZ.

Mycobacterium tuberculosis is responsible for more than 1.6 million deaths each year. One potential antibacterial target in M. tuberculosis is filamentous temperature sensitive protein Z (FtsZ), which is the bacterial homologue of mammalian tubulin, a validated cancer target. M. tuberculosis FtsZ function is essential, with its inhibition leading to arrest of cell division, elongation of the bacterial cell and eventual cell death. However, the development of potent inhibitors against FtsZ has been a challenge owing to the lack of structural information. Here we report multiple crystal structures of M. tuberculosis FtsZ in complex with a coumarin analogue. The 4-hydroxycoumarin binds exclusively to two novel cryptic pockets in nucleotide-free FtsZ, but not to the binary FtsZ-GTP or GDP complexes. Our findings provide a detailed understanding of the molecular basis for cryptic pocket formation, controlled by the conformational flexibility of the H7 helix, and thus reveal an important structural and mechanistic rationale for coumarin antibacterial activity.

Chiral liquid chromatography-tandem mass spectrometry analysis of superwarfarin rodenticide stereoisomers - Bromadiolone, difenacoum and brodifacoum - In human plasma.

Superwarfarins are second-generation long-acting anticoagulant rodenticides that can cause unintended human and wildlife toxicity due, in part, to their prolonged half-lives. Commercially available superwarfarin rodenticides are synthesized as racemates with two asymmetric carbons, producing four stereoisomers. To support studies of human plasma half-lives of individual superwarfarin stereoisomers, a method was developed based on LC-MS/MS to separate and quantify stereoisomers of the commercially important superwarfarins bromadiolone, difenacoum and brodifacoum. Human plasma samples were prepared using protein precipitation and centrifugation. Chiral-phase HPLC separation was carried out on-line with tandem mass spectrometric quantitative analysis of the eluting stereoisomers using selected-reaction monitoring with positive ion electrospray on a triple quadrupole mass spectrometer. All four stereoisomers of each superwarfarin were resolved within 12.5 min with calibration curves spanning 2-3 orders of magnitude and lower limits of quantitation between 0.87 and 2.55 ng/mL. This method was used to determine the half-lives of superwarfarin stereoisomers in plasma from patients who had inhaled synthetic cannabinoid products contaminated with superwarfarins. These data may be used to guide the development of safer next generation anticoagulant rodenticides stereoisomers.

Water vole management - Could anticoagulant rodenticides stereochemistry mitigate the ecotoxicity issues associated to their use?

Cyclic water vole population explosions can be controlled in some European countries with anticoagulant rodenticides leading sometimes to wildlife poisonings due to the toxin's tissue persistence. Here, we analyzed the pharmacokinetics of rodenticide residues in voles and we explored potential ways of improving the mass application of these agents based on the concept of stereoisomers. We demonstrated the dramatic persistence of bromadiolone in vole tissues with a hepatic half-life of about 10-30 days, while the tissue persistence of chlorophacinone is rather short with a hepatic half-life of about one day. The dramatic persistence of bromadiolone is due to the trans-isomer group (the major compound in bromadiolone), while the cis-isomer group has a short half-life. Because of resistance to chlorophacinone, the cis-bromadiolone isomers may constitute an excellent compromise between efficacy and ecotoxicological risk to control voles. A mathematical model is proposed to favor the development of baits mixed with cis-isomer groups.

Synthesis, computational study and cytotoxicity of 4-hydroxycoumarin-derived imines/enamines.

In this study, we applied a direct condensation between 3-acetyl-4-hydroxy-2H-chromen-2-one and different amines (anilines and benzyl amines) in order to synthesize some coumarin-based imines/enamines (3a-o) as cytotoxic agents. All the compounds were characterized by means of FT-IR, NMR, mass spectroscopy and elemental analyses. Since the title compounds can exist as different forms including (s-cis)-imine and (s-trans)-imine or (E and Z)-enamines, their conformational and geometrical aspects were investigated computationally by DFT method. The optimized geometry parameters, ΔE, ΔG, ΔH, Mulliken atomic charge, HOMO and LUMO energy, and NBO analysis suggested that these compounds can exist predominantly in (E)-enamine form. All the synthesized compounds (3a-o) were evaluated in vitro for their cytotoxic activities against cancer cell lines (MCF-7 and A549) and normal cell line (BEAS-2B) using the MTT assay. The 4-hydroxybenzyl derivative 3k was found to be the most potent cytotoxic agent with no selectivity, similar to doxorubicin. However, the 4-chlorobenzyl analog 3o could be considered as an equipotent compound respect to doxorubicin with higher selectivity.

Reverse Docking for the Identification of Molecular Targets of Anticancer Compounds.

Molecular docking is a useful and powerful computational method for the identification of potential interactions between small molecules and pharmacological targets. In reverse docking, the ability of one or a few compounds to bind a large dataset of proteins is evaluated in silico. This strategy is useful for identifying molecular targets of orphan bioactive compounds, proposing new molecular mechanisms, finding alternative indications of drugs, or predicting drug toxicity. Herein, we describe a detailed reverse docking protocol for the identification of potential targets for 4-hydroxycoumarin (4-HC). Our results showed that RAC1 is a target of 4-HC, which partially explains the biological activities of 4-HC on cancer cells. The strategy reported here can be easily applied to other compounds and protein datasets.

Synthesis and Evaluation of 4-Hydroxycoumarin Imines as Inhibitors of Class II Myosins.

Inhibitors of muscle myosin ATPases are needed to treat conditions that could be improved by promoting muscle relaxation. The lead compound for this study ((3-(N-butylethanimidoyl)ethyl)-4-hydroxy-2H-chromen-2-one; BHC) was previously discovered to inhibit skeletal myosin II. BHC and 34 analogues were synthesized to explore structure-activity relationships. The properties of analogues, including solubility, stability, and toxicity, suggest that the BHC scaffold may be useful for developing therapeutics. Inhibition of actin-activated ATPase activity of fast skeletal and cardiac muscle myosin II, inhibition of skeletal muscle contractility ex vivo, and slowing of in vitro actin-sliding velocity were measured. Several analogues with aromatic side arms showed improved potency (half-maximal inhibitory concentration (IC50) <1 µM) and selectivity (≥12-fold) for skeletal myosin versus cardiac myosin compared to BHC. Several analogues blocked neurotransmission, suggesting that they are selective for nonmuscle myosin II over skeletal myosin. Competition and molecular docking studies suggest that BHC and blebbistatin bind to the same site on myosin.

Porcine pancreas lipase as a green catalyst for synthesis of bis-4-hydroxy coumarins.

4-Hydroxycoumarins represents an important structural motif in life sciences molecules with remarkable biological properties. 4-Hydroxycoumarins are important precursors in the synthesis of organic compounds and critical pharmacophores in medicinal chemistry. One of the derivatives of this compound is biscoumarin, which has anticoagulant medicinal properties. Porcine pancreas lipase (PPL) has been used as an efficient green biocatalyst for the synthesis of bis-4-hydroxycoumarin compounds and expands the biocatalytic promiscuity of lipase in organic synthesis. Some aromatic aldehydes have been used in this investigation for screening and examining the ability of enzyme in knoevenagel condensation. Various parameters including temperature, solvent, and the amount of biocatalyst were investigated, ethanol, 45 °C and 10 mg of PPL gave rise to best yields. Bis-4-hydroxy coumarin compounds were synthesized under mild reaction conditions with high conversion yields (81-88%).

Mass spectrometry characterization of anticoagulant rodenticides and hydroxyl metabolites.

RATIONALE: Anticoagulant rodenticides (ARs) are used worldwide for rodent population control to protect human health and biodiversity, and to prevent agricultural and economic losses. Rodents may develop a metabolic resistance to ARs. In order to help understand such metabolic resistance, mass spectrometry was used to position the hydroxylated group of hydroxyl metabolites of second-generation ARs (SGARs). METHODS: Most AR pesticides are derived from the 4-hydroxycoumarin/thiocoumarin family. We used low-resolution and high-resolution mass spectrometry to understand the fragmentation pathways of the ARs and their respective metabolites, and to better define the structure of their tandem mass spectrometry product ions. RESULTS: Seven specific product ions were evidenced for five ARs, with their respective chemical structures. Those ions were obtained as well from the mass spectra of the hydroxyl metabolites of four SGARs, difenacoum (DFM), brodifacoum (BFM), difethialone (DFTL) and flocoumafen (FLO), with different positions of the hydroxyl group. CONCLUSIONS: The differences in chemical structure between DFM on the one hand and BFM, FLO and DFTL on the other could explain the differences in bioavailability between these two groups of molecules. The defined product ions will be used to investigate the part played by the metabolic issue in the field resistance of SGARs.

Enantiomeric fraction evaluation of the four stereoisomers of difethialone in biological matrices of rat by two enantioselective liquid chromatography tandem mass spectrometry methods: Chiral stationary phase or derivatization.

The need for the control of rodent populations with anticoagulant rodenticides remains actual, and enantioselective analytical methods are mandatory to understand ecotoxicity issues of those chiral pesticides. This study presents two enantioselective methods to achieve the residue levels and differentiated persistence of the four stereoisomers of difethialone (called in this work E1-trans, E2-cis, E3-cis and E4-trans), which is one of the most toxic second generation anticoagulant rodenticide. Their enantiomeric fraction evaluation in biological matrices of rats was determined by two LC-MS/MS methods. The first one (chiral-LC-MS/MS) combined a chiral column employed in reversed-phase mode (with acetonitrile-water mobile phase) to be compatible with mass spectrometry detection. The second one was also a LC-MS/MS method but with a reversed phase column after a derivatization step with (1S)-(-)-camphanic chloride. Extraction process combined Solid-Liquid extraction and sorbent cartridges. The methods were fully validated. The chiral column was chosen as a reference method for our laboratory because it was quicker and cheaper, and enantioresolution and sensitivity were better. This chiral-LC-MS/MS method was used to measure the enantiomeric fraction of the four stereoisomers of difethialone in rodent biological matrices (liver, plasma, blood and feces) of female rats treated with 3.5 mg/kg of difethialone. The results showed that metabolism is not the same for all the stereoisomers: cis-E3-difethialone was the most persistent, and E4-trans-difethialone was the most quickly eliminated. This chiral-LC-MS/MS method will be used to study the pharmacokinetics of the four stereoisomers of difethialone, and for ecotoxicological surveillance to evaluate the specific persistence of each stereoisomer of difethialone in case of secondary exposure of wildlife non-target species.

Efficacy of rodenticide baits with decreased concentrations of brodifacoum: Validation of the impact of the new EU anticoagulant regulation.

Anticoagulants are the most frequently used rodenticides at the global scale. Because of their persistency, bioaccumulation and potential for secondary intoxication, they have faced increasing legislative regulations. Recently, the European Union Regulation (EU) 2016/1179 resulted in the production and application of rodenticides with nearly half dose (<30 ppm) of anticoagulants. However, published data on the biological efficacy of rodenticides with decreased doses are scarce in the EU. Therefore, this work compared the efficacy of the original high-dose (50 ppm) and new low-dose (25 ppm) brodifacoum-based baits in the offspring of wild-caught house mice (Mus musculus L.). In the no-choice laboratory feeding tests, 100% animals died in all treated groups and 0% died in the control groups. The achieved time to death did not differ between the original and low-dose baits across both types of feeding trials/regimes. The low-dose baits (25 ppm) were consequently tested under field conditions in two populations showing 95.7% and 99.8% efficacy. The obtained results highlighted the good efficacy of the new baits based on low-dose brodifacoum in non-resistant mouse populations. However, further validation is required regarding the remaining anticoagulant compounds and resistant rodent populations.

In silico evaluation of pesticides as potential modulators of human DNA methyltransferases.

DNA methylations are carried out by DNA methyltransferases (DNMTs) that are key enzymes during gene expression. Many chemicals, including pesticides, have shown modulation of epigenetic functions by inhibiting DNMTs. In this work, human DNMTs were evaluated as a potential target for pesticides through virtual screening of 1038 pesticides on DNMT1 (3SWR) and DNMT3A (2QRV). Molecular docking calculations for DNMTs-pesticide complexes were performed using AutoDock Vina. Binding-affinity values and contact patterns were employed as selection criteria of pesticides as virtual hits for DNMTs. The best three DNMT-pesticides complexes selected according to their high absolute affinity values (kcal/mol), for both DNMT1 and DNMT3A, were flocoumafen (-12.5; -9.9), brodifacoum (-12.4; -8.4) and difenacoum (-12.1; -8.7). These chemicals belong to second-generation rodenticides. The most frequent predicted interacting residues for DNMT1-pesticide complexes were Trp1170A, Phe1145A, Asn1578A, Arg1574A and Pro1225A; whereas for DNMT3A those were Arg271B, Lys740A, and Glu303B. These results suggest that rodenticides used for pest control are potential DNMT ligands and therefore, may modulate DNA methylations. This finding has important environmental and clinical implications, as epigenetic pathways are critical in many biochemical processes leading to diseases.

Sodium alginate: A biopolymeric catalyst for the synthesis of novel and known polysubstituted pyrano[3,2-c]chromenes.

A library of known and new 4-aroyl-pyrano[3,2-c]chromenes have been synthesized through the one-pot, three-component reaction of 4-hydroxycoumarin, aryl glyoxals, and malononitrile or ethyl cyanoacetate in the presence of sodium alginate without any post modification, as a biopolymeric bifunctional organocatalyst, in EtOH under reflux conditions in short times. The desired products were obtained in high to excellent yields under optimized conditions. This procedure offers many advantages, such as operational simplicity, a green solvent system, short reaction times, high to excellent yields, simple work up and separation, and using a natural commercially available polysaccharide that is transition-metal-free, biodegradable and recoverable. Furthermore, sodium alginate can be recycled at least four times with negligible loss of its catalytic activity.

Synthesis of Three Bioactive Aromatic Compounds by Introducing Polyketide Synthase Genes into Engineered Escherichia coli.

Intermediates in aromatic amino acid biosynthesis can serve as substrates for the synthesis of bioactive compounds. In this study we used two intermediates in the shikimate pathway of Escherichia coli, chorismate and anthranilate, to synthesize three bioactive compounds: 4-hydroxycoumarin (4-HC), 2,4-dihydroxyquinoline (DHQ), and 4-hydroxy-1-methyl-2(1H)-quinolone (NMQ). We introduced genes for the synthesis of salicylic acid from chorismate to supply the substrate for 4-HC and the gene encoding N-methyltransferase for the synthesis of N-methylanthranilate from anthranilate. Polyketide synthases and coenzyme (Co)A ligases were tested to determine the optimal combination of genes for the synthesis of each compound. We also tested several constructs and identified the best one for increasing levels of endogenous substrates for chorismate, anthranilate, and malonyl-CoA. With the use of these strategies, 255.4 mg/L 4-HC, 753.7 mg/L DHQ, and 17.5 mg/L NMQ were synthesized. This work provides a basis for the synthesis of diverse coumarin and quinoline derivatives with potential medical applications.

The relative toxicity of brodifacoum enantiomers.

Brodifacoum (BDF) is a potent, long-acting anticoagulant rodenticide that can cause fatal poisoning in humans. The chemical structure of BDF includes 2 chiral carbons, resulting in 2 pairs of diastereomers, BDF-cis (R/S and S/R) and BDF-trans (R/R and S/S). However, the relative potency of these molecules is not known. The purpose of this study was to compare the in vitro and in vivo toxic effects of the 2 BDF diastereomer pairs. In adult Sprague-Dawley rats BDF-cis was significantly more toxic than BDF-trans (LD50 values of 219 versus 316 µg/kg, respectively) while racemic BDF had intermediate potency (266 µg/kg). In adult New Zealand white rabbits, BDF-cis had a longer half-life than BDF-trans which could contribute to its observed increased toxicity. Lastly, BDF-cis (10 µM), but not BDF-trans, damaged cultured SH-SY5Y human neuroblastoma cells by attenuating mitochondrial reductive capacity. Taken together, these data suggest that different toxic manifestations of BDF poisoning in mammals could be attributed, in part, to differences in relative enantiomer concentrations present in racemic formulations of this commercially-available toxicant.

The molecular diversity scope of 4-hydroxycoumarin in the synthesis of heterocyclic compounds via multicomponent reactions.

4-Hydroxycoumarins are some of the most versatile heterocyclic scaffolds and are frequently applied in the synthesis of various organic compounds. 4-Hydroxycoumarin-based compounds are important among heterocyclic structures due to their biological and pharmaceutical activities. In this study, we provide an overview on the recent applications of 4-hydroxycoumarin in multicomponent reactions for the synthesis of various heterocyclic compounds during the time period of 2015-2018.

Effective degradation of refractory nitrobenzene in water by the natural 4-hydroxycoumarin under solar illumination.

In this study, nitrobenzene (NB) as typical refractory organic pollutants was effectively degraded by a new green approach, which was achieved by the chemical effect of natural organic matters (NOMs) under solar illumination as a potential natural degradation process. 4-hydroxycoumarin (4HC), which is natural compound and can be extracted from many plants, was found as an efficient photosensitizer to promote the photoreduction of NB to 4-aminophenol under the solar illumination. The reaction products were definitely identified by LC-MS/MS and 1H NMR. The response spectrum of 4HC excited state (4H-chromene-2,4-diol radical, S1) as key intermediate was also obtained by transient absorption spectroscopy (TAS) measurements, which showed that the decay time of S1 was around 250 ps. Then, the measurements of electron paramagnetic resonance (EPR) confirmed the existence of OH. As a result, the reaction mechanism between 4HC and NB was proposed. In addition, the influence parameters such as light sources, gas surroundings, solvents, pH values were investigated to further reveal the reaction process.